KR20210002260A - A method of producing sulfur-containing amino acids and derivatives thereof - Google Patents

Abstract

The present application relates to a method for manufacturing a sulfur-containing amino acid or a sulfur-containing amino acid derivative, which comprises culturing a microorganism in which the protein activity encoded by a ssuD gene is enhanced compared to the intrinsic activity in a medium containing thiosulfate.

Description

A method of producing sulfur-containing amino acids and derivatives thereof

The present application relates to a method for preparing a sulfur-containing amino acid or a sulfur-containing amino acid derivative, comprising culturing a microorganism in which the protein activity encoded by the ssuD gene is enhanced compared to the intrinsic activity in a medium containing thiosulfate.

L-amino acids are industrially produced by a fermentation method using microorganisms belonging to the genus Brevibacterium, the genus Corynebacterium, and the genus Escherichia. In this production method, strains isolated from nature, artificial mutants of the strain, or microorganisms that have been mutated to increase the activity of enzymes involved in L-amino acid biosynthesis by recombinant DNA technology are used.

On the other hand, sulfur-containing amino acids are used as animal feeds, food additives, pharmaceutical solutions, and synthetic raw materials for pharmaceuticals, and studies have been made to biologically produce such sulfur-containing amino acids and derivatives thereof.

For example, in German Laid-Open Patent Publication DE 10,305,774 A1, the metJ gene on the genomic gene of Escherichia coli was removed, and the L-methionine-releasing factor YjeH protein was over-expressed to 0.8 g/ It was reported that L-methionine was produced. In addition, BrnF and BrnE polypeptides, which are L-methionine-releasing factors of Corynebacterium glutamicum, were reported (C. Troschel, et al, Journal of Bacteriology, p.3786-3794, June 2005).

On the other hand, in producing sulfur-containing amino acids, the amount of NADPH consumed in microorganisms varies according to the reducing power of the sulfur source. For example, since sulfide does not require NADPH, the yield is theoretically the highest, and since sulfate requires four NADPH, the yield is theoretically low. However, since sulfide is known to cause cell damage, it has a disadvantage of low stability. Therefore, when NADPH demand is low, and as a source of sulfur with high intracellular stability, a high production yield can be expected when thiosulfate is used for the production of sulfur-containing amino acids. However, there are insufficient studies that can be used efficiently in microorganisms in Corynebacterium.

Under this background, the present inventors newly identified that the protein encoded by the ssuD gene is involved in the use of thiosulfate, and by confirming that the microorganism having enhanced its activity uses thiosulfate as a sulfur source, the ability to produce sulfur-containing amino acids is enhanced. The present invention has been completed.

It is an object of the present application to provide a method for producing a sulfur-containing amino acid or a sulfur-containing amino acid derivative, comprising culturing a microorganism in which the protein activity encoded by the ssuD gene is enhanced compared to the intrinsic activity in a medium containing thiosulfate. .

Another object of the present application is to provide a microorganism for the production of a sulfur-containing amino acid or a sulfur-containing amino acid derivative in which the protein activity encoded by the ssuD gene is enhanced compared to the intrinsic activity.

Another object of the present application is a microorganism, or a culture thereof, in which the protein activity encoded by the ssuD gene is enhanced compared to the intrinsic activity; And it is to provide a composition for preparing a sulfur-containing amino acid or a sulfur-containing amino acid derivative comprising thiosulfate.

This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present application may be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present application belong to the scope of the present application. In addition, it cannot be considered that the scope of the present application is limited by the specific description described below.

Further, those of ordinary skill in the art may recognize or ascertain a number of equivalents to certain aspects of the present application described in this application using only routine experimentation. Also, such equivalents are intended to be included in this application.

One aspect of the present application provides a method for preparing a sulfur-containing amino acid or a sulfur-containing amino acid derivative comprising culturing a microorganism in which the protein activity encoded by the ssuD gene is enhanced compared to the intrinsic activity in a medium containing thiosulfate. do. The present application provides a method for preparing a sulfur-containing amino acid or a sulfur-containing amino acid derivative, comprising culturing a genetically modified microorganism in a medium containing thiosulfate, wherein the microorganism is compared with the microorganism before the genetic modification, the ssuD gene It may include a genetic modification that increases the activity of the protein encoded by. In one embodiment of the present application, the method may be a method of increasing the production of a sulfur-containing amino acid or a sulfur-containing amino acid derivative.

The manufacturing method may include contacting a microorganism or a culture thereof with a thiosulfate whose protein activity encoded by the ssuD gene is enhanced compared to the intrinsic activity.

The present application is characterized by a new discovery that the protein encoded by the ssuD gene is involved in the use of thiosulfate.

In the present application, the'protein encoded by the ssuD gene' is a protein encoded by the ssuD gene, or a protein expressed by the ssuD gene, and may be referred to as a'SsuD protein' (hereinafter referred to as a "SsuD protein") Conventionally, the SsuD protein is known as a protein that degrades sulfonate (R-SO ₃ ), specifically, as one of alkanesulfonate monooxygenase, as a sulfonate monooxygenase. However, it is not known whether the SsuD protein is involved in the use of thiosulfate rather than organic sulfates.

In the present application, it was newly identified that the SsuD protein is also involved in the use of thiosulfate, specifically, it performs a function as a reductase that reduces thiosulfate, and by enhancing the activity of the SsuD protein, the production of sulfur-containing amino acids It was confirmed that can be increased. Accordingly, an embodiment of the present application provides the use of SsuD protein for the production of sulfur-containing amino acids using thiosulfate. The SsuD protein can be used as a thiosulfate reductase. In addition, an embodiment of the present application provides the use of a microorganism for producing a sulfur-containing amino acid or a sulfur-containing amino acid derivative, or a microorganism for producing a sulfur-containing amino acid or a sulfur-containing amino acid derivative, having enhanced SsuD protein activity. In addition, another embodiment of the present application is a microorganism, or a culture thereof, in which the SsuD protein activity is enhanced compared to the intrinsic activity; And it provides a composition for preparing a sulfur-containing amino acid or a sulfur-containing amino acid derivative comprising thiosulfate.

The SsuD protein of the present application may be a protein encoded by the ssuD gene, which may be referred to as'SsuD protein'or'thiosulfatereductase', or may be referred to as'alkanesulfonate monooxygenase' known in the art. . In one embodiment, the SsuD protein of the present application may be'sulfonate monooxygenase'or'thiosulfatereductase' derived from the genus Corynebacterium, and specifically, LLM class flavin derived from the genus Corynebacterium It may be a protein named -dependent oxidoreductase, and more specifically, it may be a SsuD protein derived from Corynebacterium glutamicum, but is not limited thereto. The sequence of the SsuD protein can be identified in a known database such as NCBI.

In one embodiment, the SsuD protein comprises the amino acid sequence of SEQ ID NO: 43, or at least 80%, 90%, 92%, 94%, 95%, 96%, 97%, 98 with the amino acid sequence of SEQ ID NO: 43 % Or 99% homology or identity. In addition, if an amino acid sequence that has the homology or identity and exhibits efficacy corresponding to the polypeptide, it is obvious that some sequences are included within the scope of the present application even if they have an amino acid sequence that has been deleted, modified, substituted or added.

In addition, as a probe that can be prepared from a known gene sequence, for example, a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions with a complementary sequence to all or part of the nucleotide sequence encoding the polypeptide, A polypeptide having a sulfonate monooxygenase activity and a thiosulfate reducing activity may be included without limitation.

That is, in the present application,'protein or polypeptide comprising an amino acid sequence described by a specific sequence number','a protein or polypeptide consisting of an amino acid sequence described by a specific sequence number' or'a protein having an amino acid sequence described by a specific sequence number, or Even if it is described as'polypeptide', if it has the same or corresponding activity as a polypeptide consisting of the amino acid sequence of the corresponding sequence number, a protein having an amino acid sequence in which some sequence is deleted, modified, substituted, conservatively substituted or added is also It is obvious that it can be used in this application. For example, if the amino acid sequence N-terminus and/or C-terminus has a sequence that does not change the function of the protein, a naturally occurring mutation, a silent mutation or a conservative substitution thereof. .

The "conservative substitution" refers to the substitution of one amino acid for another amino acid having similar structural and/or chemical properties. Such amino acid substitutions can generally occur based on similarity in the polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; Negatively charged (acidic) amino acids include glutamic acid and aspartic acid; Aromatic amino acids include phenylalanine, tryptophan and tyrosine, and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine and tryptophan.

The SsuD protein may be encoded by the polynucleotide sequence of SEQ ID NO: 8, but is not limited thereto.

In the present application, the term "polynucleotide" has the meaning of comprehensively including DNA or RNA molecules, and nucleotides, which are basic structural units in polynucleotides, may include not only natural nucleotides but also analogs with modified sugar or base moieties ( Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)).

The polynucleotide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homologous to the polynucleotide encoding the SsuD protein of the present application or the SsuD protein of the present application. Or it may be a polynucleotide encoding a polypeptide having identity. Specifically, the polynucleotide encoding a protein comprising an amino acid sequence having at least 80% homology or identity to the amino acid sequence of SEQ ID NO: 43 is at least 80%, at least 80%, 90%, 92 with the nucleotide sequence of SEQ ID NO: 8 %, 94%, 95%, 96%, 97%, 98% or 99% homology or identity can be a polynucleotide.

In addition, it is apparent that a polynucleotide that can be translated into a protein comprising an amino acid sequence having 80% or more homology or identity to SEQ ID NO: 43 by codon degeneracy may also be included. Or a probe that can be prepared from a known gene sequence, for example, an amino acid having at least 80% identity with the amino acid sequence of SEQ ID NO: 43 by hydride under stringent conditions with a complementary sequence for all or part of the base sequence. Any polynucleotide sequence encoding a protein comprising the sequence may be included without limitation. The "stringent conditions" refer to conditions that allow specific hybridization between polynucleotides. These conditions are described in, e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology , John Wiley & Sons, Inc., New York). For example, between genes with high homology or identity, 70% or more, 80% or more, specifically 85% or more, specifically 90% or more, more specifically 95% or more, more specifically 97% or more , In particular, a condition in which genes having 99% or more homology or identity are hybridized, and genes with lower homology or identity are not hybridized, or a washing condition of ordinary southern hybridization at 60°C, 1× SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, more specifically 68°C, 0.1×SSC, at a salt concentration and temperature corresponding to 0.1% SDS, once, specifically 2 Conditions for washing times to three times can be listed.

Hybridization requires that two polynucleotides have a complementary sequence, although a mismatch between bases is possible depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases capable of hybridizing to each other. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Thus, the present application may also include substantially similar polynucleotide sequences as well as isolated polynucleotide fragments that are complementary to the entire sequence.

Specifically, polynucleotides having homology or identity can be detected using hybridization conditions including a hybridization step at a Tm value of 55° C. and using the above-described conditions. Further, the Tm value may be 60°C, 63°C, or 65°C, but is not limited thereto and may be appropriately adjusted by a person skilled in the art according to the purpose.

The appropriate stringency to hybridize a polynucleotide depends on the length and degree of complementarity of the polynucleotide, and the parameters are well known in the art.

In the present application, the term "homology" or "identity" refers to the degree to which two given amino acid sequences or base sequences are related and may be expressed as a percentage. The terms homology and identity can often be used interchangeably.

The sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard alignment algorithms, and the default gap penalty established by the program used can be used together. Substantially, homologous or identical sequences are generally at least about 50%, 60%, 70%, 80% of the sequence full or full-length in medium or high stringent conditions. Or it can hybridize to 90% or more. Hybridization is also contemplated for polynucleotides containing degenerate codons instead of codons in the polynucleotide.

Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined, for example, in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: Can be determined using a known computer algorithm such as the "FASTA" program using default parameters as in 2444. Alternatively, as performed in the Needleman program (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) of the EMBOSS package, Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) can be used to determine. (GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215] : 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO ETA/.] (1988) SIAM J Applied Math 48: 1073) For example, homology, similarity or identity can be determined using BLAST, or ClustalW of the National Center for Biotechnology Information.

The homology, similarity or identity of a polynucleotide or polypeptide can be found in, for example, Smith and Waterman, Adv. Appl. As known in Math (1981) 2:482, for example, Needleman et al. (1970), J Mol Biol. 48: 443 can be determined by comparing sequence information using a GAP computer program. In summary, the GAP program defines the total number of symbols in the shorter of two sequences, divided by the number of similarly aligned symbols (ie, nucleotides or amino acids). The default parameters for the GAP program are (1) a monolithic comparison matrix (contains values of 1 for identity and 0 for non-identity) and Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. As disclosed by 353-358 (1979), Gribskov et al (1986) Nucl. Acids Res. 14: weighted comparison matrix of 6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); And (3) no penalty for end gaps. Thus, as used herein, the term “homology” or “identity” refers to the relevance between sequences.

The "enhancing or increasing the activity of the protein encoded by the ssuD gene or the activity of the SsuD protein" may be referred to as a genetic modification in which the activity of the protein encoded by the ssuD gene is increased. It can mean what has been done.

In the present application, the term "the activity of a protein is enhanced relative to the intrinsic activity" may also be expressed as "increase in activity", and prior to a genetic modification microorganism or non-modified or unmodified) means that the intrinsic activity of the protein possessed by the microorganism is improved compared to the activity before modification. In the present application, the term "intrinsic" refers to a condition originally possessed by the parent strain before the change in the trait when the trait of a microorganism is changed due to genetic variation caused by natural or artificial factors. The increase in activity may include both introduction of a foreign protein and enhancement of the intrinsic protein activity. Increasing/enhancing the activity of the protein can be achieved by increasing/enhancing the expression of a gene.

Specifically, the activity enhancement in this application,

1) increase the number of copies of the polynucleotide encoding the protein,

2) modification of the expression control sequence to increase the expression of the polynucleotide,

3) modification of the polynucleotide sequence on the chromosome to enhance the activity of the protein,

4) introduction of a foreign polynucleotide exhibiting the activity of the protein or a codon-optimized variant polynucleotide of the polynucleotide, or

5) It may be performed by a method of transforming to be strengthened by a combination thereof, but is not limited thereto.

The 1) increase in the copy number of the polynucleotide may be performed in a form operably linked to a vector, although not particularly limited thereto, or may be performed by being inserted into a chromosome in a host cell. Specifically, the polynucleotide encoding the protein of the present invention is operably linked to a vector capable of replicating and functioning independently of the host and introduced into a host cell, or the polynucleotide is inserted into a chromosome in the host cell. The polynucleotide is operably linked to a vector capable of being introduced into a host cell, thereby increasing the number of copies of the polynucleotide in the chromosome of the host cell.

Next, 2) modification of the expression control sequence to increase the expression of the polynucleotide is not particularly limited thereto, but deletion, insertion, non-conservative or conservative substitution of the nucleic acid sequence to further enhance the activity of the expression control sequence, or It may be performed by inducing a mutation in the sequence by a combination of, or by replacing with a nucleic acid sequence having a stronger activity. The expression control sequence, although not particularly limited thereto, may include a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence controlling termination of transcription and translation, and the like.

A strong heterologous promoter may be connected to the top of the polynucleotide expression unit instead of the original promoter. Examples of the strong promoter include the CJ7 promoter (Korean Patent No. 0620092 and WO2006/065095), the lysCP1 promoter (WO2009/096689), and spl1. Promoter, spl7 promoter, spl13 promoter (Korean Patent No. 1783170), gapA promoter, EF-Tu promoter, groEL promoter, aceA or aceB promoter, but are not limited thereto. In addition, 3) modification of the polynucleotide sequence on the chromosome is not particularly limited thereto, but the expression control sequence by deletion, insertion, non-conservative or conservative substitution of the nucleic acid sequence to further enhance the activity of the polynucleotide sequence, or a combination thereof It can be carried out by inducing a phase mutation, or by replacing with an improved polynucleotide sequence to have a stronger activity.

In addition, 4) introduction of a foreign polynucleotide sequence may be performed by introducing a foreign polynucleotide encoding a protein exhibiting the same/similar activity as the protein, or a codon-optimized variant polynucleotide thereof into a host cell. The foreign polynucleotide may be used without limitation in its origin or sequence as long as it exhibits the same/similar activity as the protein. In addition, the introduced foreign polynucleotide can be introduced into the host cell by optimizing its codon so that optimized transcription and translation are performed in the host cell. The introduction may be performed by appropriately selecting a known transformation method by a person skilled in the art, and the introduced polynucleotide may be expressed in a host cell to produce a protein, thereby increasing its activity.

Finally, 5) the method of modifying to be enhanced by the combination of 1) to 4) above, includes an increase in the copy number of the polynucleotide encoding the protein, modification of the expression control sequence to increase its expression, and the polynucleotide on the chromosome Modification of the sequence and the modification of foreign polynucleotides or codon-optimized variant polynucleotides exhibiting the activity of the protein may be applied together.

The term "vector" of the present application refers to a DNA preparation containing a polynucleotide sequence encoding the protein of interest operably linked to a suitable regulatory sequence so that the protein of interest can be expressed in a suitable host. The regulatory sequence may include a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling termination of transcription and translation. Vectors can be transformed into a suitable host cell and then replicated or function independently of the host genome, and can be integrated into the genome itself. For example, a polynucleotide encoding a target protein in a chromosome may be replaced with a mutated polynucleotide through a vector for intracellular chromosome insertion. Insertion of the polynucleotide into the chromosome may be performed by any method known in the art, for example, homologous recombination, but is not limited thereto.

The vector of the present application is not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used as a phage vector or a cosmid vector, and as a plasmid vector, pBR system, pUC system, pBluescriptII system , pGEM system, pTZ system, pCL system, pET system, etc. can be used. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, and the like can be used.

The term "transformation" in the present application means introducing a vector containing a polynucleotide encoding a target protein into a host cell so that the protein encoded by the polynucleotide can be expressed in the host cell. Transformed polynucleotides may include all of them, whether inserted into the chromosome of the host cell or located outside the chromosome, as long as it can be expressed in the host cell. In addition, the polynucleotide includes DNA and RNA encoding the target protein. The polynucleotide may be introduced in any form as long as it can be introduced into a host cell and expressed. For example, the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct containing all elements necessary for self-expression. The expression cassette may generally include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of an expression vector capable of self-replicating. In addition, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell, but is not limited thereto.

In addition, the term "operably linked" in the present application means that the gene sequence is functionally linked to a promoter sequence that initiates and mediates transcription of a polynucleotide encoding a protein of interest herein.

The method of transforming the vector of the present application includes any method of introducing a nucleic acid into a cell, and may be performed by selecting an appropriate standard technique as known in the art depending on the host cell. For example, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl ₂ ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and acetic acid There is a lithium-DMSO method and the like, but is not limited thereto.

The microorganism may be a microorganism that produces L-amino acid.

The term "microorganism producing L-amino acid" in the present application refers to a microorganism having the ability to produce L-amino acid naturally, or a microorganism to which the ability to produce L-amino acid is imparted to a parent strain without the ability to produce L-amino acid. For example, the microorganism producing the L-amino acid may be a microorganism whose amino acid biosynthetic pathway is enhanced or the degradation pathway is weakened. For example, the microorganism producing L-amino acid may be a microorganism producing L-methionine, and may be a microorganism having an enhanced L-methionine biosynthesis pathway. For example, the L-amino acid-producing microorganism has reduced or inactivated the activity of McbR (methionine and cysteine biosynthesis repressor protein) or MetJ protein, or the activity of methionine synthase (MetH) or sulfite reductase (CysI). It may be a microorganism to which the methionine-producing ability is enhanced and/or added by the enhancement. Alternatively, it may be a microorganism in which the expression of a gene encoding an enzyme in other L-amino acid biosynthetic pathways is enhanced or an enzyme in the degradation pathway is weakened/inactivated.

Specifically, examples of proteins or genes capable of regulating expression to enhance L-amino acid biosynthetic pathways or weaken/inactivate degradation pathways are as follows. Proteins, representative genes encoding proteins, and representative EC numbers were listed in order. The first letter of the protein was capitalized, and the gene was written in italics. For example, thiosulphate sulphurtransferases such as Rdl2p, GlpE, PspE, YgaP, ThiI, YbbB, SseA, YnjE, YceA, YibN, NCgl0671, NCgl1369, NCgl2616, NCgl0053, NCgl0054, NCGl2678, NCgl2890; Sulfite reductase, cysI ; Thiosulphate /sulphate transport system, cysPUWA (EC 3.6.3.25); 3'-phosphoadenosine 5''-phosphosulphate reductase, cysH (EC 1.8.4.8); Sulphite reductase, cysJI (EC 1.8.1.2); Cysteine synthase A, cysK (EC 2.5.1.47); Cysteine synthase B, cysM (EC 2.5.1.47); Serine acetyltransferase, cysE (EC 2.3.1.30); Glycine cleavage system, gcvTHP-lpd (EC 2.1.2.10, EC 1.4.4.2, EC 1.8.1.4); Lipoyl synthase, lipA (EC 2.8.1.8); Lipoyl protein ligase, lipB (EC 2.3.1.181); Phosphoglycerate dehydrogenase, serA (EC 1.1.1.95); 3-phosphoserine phosphatase, serB (EC 3.1.3.3); 3-phosphoserine/phosphohydroxythreonine aminotransferase, serC (EC 2.6.1.52); Serine hydroxymethyltransferase, glyA (EC 2.1.2.1); Aspartokinase I (EC 2.7.2.4); Homoserine dehydrogenase I, thrA (EC 1.1.1.3); Aspartate kinase, lysC (EC 2.7.2.4); Homoserine dehydrogenase, hom (EC 1.1.1.3); Homoserine O-acetyltransferase, metX (EC 2.3.1.31); Homoserine O-succinyltransferase, metA (EC 2.3.1.46); Cystathionine gamma-synthase, metB (EC 2.5.1.48); β-CS-lyase, aecD (EC 4.4.1.8, beta-lyase); Cystathionine beta-lyase, metC (EC 4.4.1.8); B12-independent homocysteine S-methyltransferase, metE (EC 2.1.1.14); Methionine synthase, metH (EC 2.1.1.13); Methylenetetrahydrofolate reductase, metF (EC 1.5.1.20); L-methionine exocarrier BrnFE; Valine transporter YgaZH (B2682, B2683), ygaZH (b2682. b2683 ); External transporter YjeH, b4141 ; Pyridine nucleotide transhydrogenase PntAB, pntAB (EC 1.6.1.2); O-succinylhomoserine sulfhydrylase, MetZ (EC 2.5.1.48); And one or more proteins selected from among phosphoenolpyruvate carboxylase and Pyc (EC 4.1.1.31) or enhancing the activity of some proteins constituting the system or overexpressing the polynucleotide encoding the L-amino acid It can enhance biosynthetic pathways or weaken degradation pathways. Alternatively, glucose 6-phosphate isomerase, pgi (EC 5.3.1.9); Homoserine kinase, thrB (EC 2.7.1.39); S-adenosylmethionine synthase, metK (EC 2.5.1.6); Dihydrodipicolinate synthase, dapA (EC 4.2.1.52); Phosphoenolpyruvate carboxylkinase, pck (EC 4.1.1.49);, formyltetrahydrofolate hydrolase, purU (EC 3.5.1.10); Pyruvate kinase I, pykF (EC 2.7.1.40); Pyruvate kinase II, pykA (EC 2.7.1.40); Cystathionine γ-lyase, cg3086 (EC 4.4.1.1); Cystathionine β-synthesizing enzyme, cg2344 (EC 4.2.1.22); Regulatory proteins Cg3031, cg3031 ; Methionine and cysteine biosynthesis repressor protein McbR, mcbR ; L-methionine synthesis transcriptional regulator (Met transcriptional repressor protein), metJ ; L-methionine transporters MetQNI, metQ, metN, metI ; N-acyltransferase, yncA ; sRNA fnrS ; And L-methionine transporter, the activity of one or more proteins selected from the group consisting of metP may be inactivated or attenuated, or the expression of the gene encoding the protein may be suppressed or eliminated.

However, this is only an example, and may be a microorganism in which expression of a gene encoding an enzyme in various known L-amino acid biosynthetic pathways is enhanced or an enzyme in a degradation pathway is weakened/inactivated, but is not limited thereto. Microorganisms that produce the L-amino acids can be prepared by applying various known methods.

The term "weak/inactivate protein activity" in the present application means that the expression of an enzyme or protein is not expressed at all compared to the natural wild-type strain, the parent strain, or the strain in which the corresponding protein is unmodified, or there is no activity. It means reduced. In this case, the decrease is when the activity of the protein is decreased compared to the activity of the protein originally possessed by the microorganism due to mutation of the gene encoding the protein, modification of the expression control sequence, or deletion of a part or all of the gene, and the gene encoding it In the case where the overall protein activity level in the cell is lower than that of the native strain or the strain before transformation due to inhibition of expression of or translation inhibition, the concept includes a combination thereof. In the present application, the inactivation/weakening may be achieved by applying various methods well known in the art. Examples of the method include: 1) a method of deleting all or part of the gene encoding the protein; 2) modification of the expression control sequence to reduce the expression of the gene encoding the protein, 3) modification of the gene sequence encoding the protein so that the activity of the protein is removed or weakened, 4) the gene encoding the protein Introduction of antisense oligonucleotides (eg, antisense RNA) that complementarily bind to the transcript of 5) A secondary structure is formed by adding a sequence complementary to the sine-Dalgarno sequence to the front of the sine-Dalgarno sequence of the gene encoding the protein, thereby making it impossible to attach a ribosome. Way; 6) There is a method of adding a promoter transcribed in the opposite direction to the 3'end of the ORF (open reading frame) of the polynucleotide sequence of the gene encoding the protein (Reverse transcription engineering, RTE), etc., and a combination thereof Also can be achieved, but is not particularly limited thereto, and can be applied by appropriately selecting an inactivation method known in the art.

The microorganism may be Corynebacterium sp., Escherichia sp., or Lactobacillus sp., but is not limited thereto. For the purposes of the present application, the microorganism may be included without limitation as long as the intrinsic activity of the SsuD protein is enhanced or the production capacity of L-amino acids and/or derivatives thereof is increased by the introduction of the foreign SsuD protein.

The L-amino acids and/or derivatives thereof may be sulfur-containing amino acids and/or derivatives of sulfur-containing amino acids.

The "Corynebacterium genus microorganism" of the present application may include all Corynebacterium genus microorganisms. Specifically, Corynebacterium glutamicum ( Corynebacterium glutamicum ), Corynebacterium crudilactis ( Corynebacterium crudilactis ), Corynebacterium deserti ( Corynebacterium deserti ), Corynebacterium Epiciens ( Corynebacterium efficiens ) , Corynebacterium callunae , Corynebacterium stationis , Corynebacterium singulare , Corynebacterium halotolerans , Corynebacterium Corynebacterium striatum , Corynebacterium ammoniagenes , Corynebacterium pollutisoli , Corynebacterium imitans , Corynebacterium testudino Lys ( Corynebacterium testudinoris ) or Corynebacterium flavescens ( Corynebacterium flavescens ) may be, more specifically Corynebacterium glutamicum ( Corynebacterium glutamicum ), Corynebacterium calunae ( Corynebacterium callunae ), Coryne Bacterium crenatum ( Corynebacterium crenatum ), or Corynebacterium deserti ( Corynebacterium deserti ).

The "Escherichia genus microorganism" in the present application may include all Escherichia genus microorganisms. Specifically, it may be Escherichia coli , but is not limited thereto.

On the other hand, it has already been known that microorganisms of the genus Corynebacterium or genus Escherichia can produce L-amino acids, but their production capacity is remarkably low, and all genes or mechanisms acting on the production mechanism have not been identified. . Therefore, the'microorganism producing L-amino acid' of the present application is a microorganism that has improved L-amino acid production ability by enhancing or weakening/inactivating the activity of the natural wild-type microorganism itself, a gene related to the L-amino acid production mechanism, Or it refers to a microorganism that has an improved L-amino acid production ability by introducing or enhancing the activity of an external gene. The culture of the microorganism of the present application may be prepared by culturing the microorganism of the present application in a medium.

The term "cultivation" in the present application means growing the microorganism under appropriately controlled environmental conditions. The cultivation process of the present application may be performed according to a suitable medium and culture conditions known in the art. This culture process can be easily adjusted and used by those skilled in the art according to the selected strain. The step of culturing the microorganism is not particularly limited thereto, but may be performed by a known batch culture method, a continuous culture method, a fed-batch culture method, or the like. At this time, the culture conditions are not particularly limited thereto, but a basic compound (eg, sodium hydroxide, potassium hydroxide or ammonia) or an acidic compound (eg, phosphoric acid or sulfuric acid) is used to provide an appropriate pH (eg, pH 5 to 9, specifically PH 7 to 9) can be adjusted. In addition, during cultivation, an antifoaming agent such as fatty acid polyglycol ester can be used to suppress the generation of air bubbles. In addition, in order to maintain the aerobic state of the culture, oxygen or oxygen-containing gas is injected into the culture, or the anaerobic and microaerobic state To maintain, nitrogen, hydrogen or carbon dioxide gas may be injected without injection of gas. The culture temperature may be maintained at 25 ℃ to 40 ℃, specifically 30 ℃ to 37 ℃, but is not limited thereto. The cultivation period may be continued until the production amount of the desired useful substance is obtained, and specifically, the cultivation may be performed for about 0.5 to 60 hours, but is not limited thereto. In addition, the culture medium used is a carbon source such as sugars and carbohydrates (e.g. glucose, sucrose, lactose, fructose, maltose, molase, starch and cellulose), fats and fats (e.g., soybean oil, sunflower seeds). Oil, peanut oil and coconut oil), fatty acids (such as palmitic acid, stearic acid and linoleic acid), alcohols (such as glycerol and ethanol), and organic acids (such as acetic acid) can be used individually or in combination. , Is not limited thereto. Nitrogen sources include nitrogen-containing organic compounds (e.g. peptone, yeast extract, broth, malt extract, corn steep liquor, soybean meal and urea), or inorganic compounds (e.g. ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate) The etc. may be used individually or may be used in combination, but is not limited thereto. Potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and a sodium-containing salt corresponding thereto may be used individually or in combination as a source of phosphorus, but are not limited thereto. In addition, the medium may contain essential growth-promoting substances such as other metal salts, amino acids and vitamins.

The term "suphur source" in the present application is used interchangeably with "sulfur source" and refers to a material containing elemental sulfur that can be used for the production of sulfur-containing amino acids.

In culturing microorganisms, sulfur source may be an important factor determining metabolic reaction pathways in microorganisms. However, for various sources of sulfur, it is not clear which factor carries it and which factor decomposes it. For example, it is known that wild-type Corynebacterium glutamicum can use various sources of sulfur, but among them, SsuABC protein is not involved in the transport of sulfate or sulfite, and is known to be involved in the transport of aliphatic sulfonate. There is only. (DJ Koch, C. Ruckert, DA Rey, A. Mix, A. Puhler, J. Kalinowski. 2005. Role of the ssu and seu Genes of Corynebacterium glutamicum ATCC 13032 in Utilization of Sulfonates and Sulfonate Esters as Sulfur Sources. AEM. 71.10.6104-6114.2005), that is, a protein that carries sulfur sources into cells has substrate specificity. In addition, even after the sulfur source is transported into the cell, the enzyme that decomposes it is also different depending on the structure and functional groups of the sulfur source, and the metabolic pathway using the sulfur source may also vary. For example, when sulfate is used as a sulfur source, CysZ carries it, and it is known that CysDN, CysH, and CysI are involved until sulfide formation. (Bolten, Christoph J., Hartwig Schroder, Jeroen Dickschat, and Christoph Wittmann. Towards Methionine Overproduction in Corynebacterium glutamicum Methanethiol and Dimethyldisulfide as Reduced Sulfur Sources. J. Microbiol. Biotechnol. (2010), 20(8), 1196-1203) However, in the production of sulfur-containing amino acids, when thiosulfate is used as a sulfur source, which factors transport and decompose it has not been clearly identified. The present application is significant in identifying a protein using thiosulfate.

For the purposes of this application, the sulfur source may mean thiosulfate. In the present application, the sulfur source may specifically include a thiosulfate such as ammonium thiosulfate or sodium thiosulfate, or a reduced raw material such as sulfite, H ₂ S, sulfide, sulfide derivative, methyl mercaptan, thio It may be in the form of a mixture of thiosulfate with organic and inorganic sulfur-containing compounds such as glycolite, thiocyanate and thiourea. Alternatively, substances other than thiosulfate may not be included as a sulfur source.

The term "sulfur-containing amino acid" or "sulfur-containing amino acid derivative" in the present application refers to an amino acid containing elemental sulfur or a derivative thereof, specifically, methionine, cysteine, cystine, and lanthionine (lanthionine), homocysteine (homocysteine), homocystine (homocystine), homolanthionine (homolanthionine), and may be any one selected from taurine (taurine), but the scope of the present application if an amino acid containing sulfur and a derivative thereof Included without limitation.

The method may include recovering a sulfur-containing amino acid or a sulfur-containing amino acid derivative from the microorganism or culture medium.

The recovering step is a desired sulfur-containing amino acid or a sulfur-containing amino acid derivative from the medium using a suitable method known in the art according to the method of culturing the microorganism of the present application, for example, a batch, continuous, or fed-batch culture method. Can be recovered. For example, centrifugation, filtration, treatment with a crystallized protein precipitant (salt-in method), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography Various chromatography such as, HPLC, and methods thereof may be used in combination, but are not limited to these examples.

The recovery step may include an additional purification process. The purification process can use a suitable method known in the art.

Another aspect of the present application provides a microorganism for producing a sulfur-containing amino acid or a sulfur-containing amino acid derivative in which the protein activity encoded by the ssuD gene is enhanced compared to the intrinsic activity.

Another aspect of the present application is a microorganism, or a culture thereof, in which the protein activity encoded by the ssuD gene is enhanced compared to the intrinsic activity; And it provides a composition for preparing a sulfur-containing amino acid or a sulfur-containing amino acid derivative comprising thiosulfate.

Proteins, microorganisms, cultures, thiosulfate and sulfur-containing amino acids encoded by the ssuD gene are as described above.

The composition may further include an optional component capable of assisting in preparing a sulfur-containing amino acid or a sulfur-containing amino acid derivative, and such components can be appropriately selected from those known in the art.

By using the microorganism, composition of the present application, and the method for preparing sulfur-containing amino acid or sulfur-containing amino acid using the same, a sulfur-containing amino acid or a derivative thereof can be mass-produced, making it useful for the production of useful products including sulfur-containing amino acids or derivatives thereof. Can be used.

Hereinafter, the present application will be described in more detail through examples and experimental examples. However, these Examples and Experimental Examples are for illustrative purposes only, and the scope of the present application is not limited to these Examples and Experimental Examples.

Example 1: mcbR Production of synthetic vector for gene deletion

First, in order to prepare a methionine-producing strain, a representative sulfur-containing amino acid, mcbR (J. Biotechnol. 103:51-65) has a Corynebacterium glutamicum ATCC13032 strain and encodes a known methionine cysteine transcriptional regulator protein (J. Biotechnol. 103:51-65). , 2003) A vector was constructed for inactivation of.

Specifically, a recombinant plasmid vector was constructed by the following method in order to delete the mcbR gene on the Corynebacterium ATCC13032 chromosome.

Based on the nucleotide sequence reported to the National Institutes of Health (NIH Genbank), the mcbR gene and peripheral sequence (SEQ ID NO: 1) of Corynebacterium glutamicum were obtained.

Using the chromosomal DNA of Corynebacterium glutamicum ATCC 13032 as a template, PCR was performed using the primers of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. PCR conditions were denatured at 95° C. for 5 minutes, then denaturation at 95° C. for 30 seconds, annealing at 53° C. for 30 seconds, and polymerization at 72° C. for 30 seconds were repeated 30 times, followed by polymerization at 72° C. for 7 minutes. As a result, each 700bp DNA fragment was obtained.

In Corynebacterium glutamicum, the pDZ vector (Korean Patent Registration No. 10-0924065) and the amplified mcbR gene fragments were treated with SmaI restriction enzyme for chromosome introduction, and after isothermal assembly cloning reaction, E. coli DH5α was transformed and plated on LB solid medium containing kanamycin (25mg/ℓ). After selecting a colony transformed with a vector into which the fragments of the target genes were deleted through PCR, a plasmid was obtained using a plasmid extraction method, and was named pDZ-ΔmcbR.

Example 2: mcbR Creation and cultivation of strains with defective genes

The pDZ-ΔmcbR vector prepared in Example 1 was transformed into ATCC13032 strains by electroporation, respectively, by homologous recombination on chromosomes (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). . Then, secondary recombination was performed in a solid medium containing sucrose. The strain in which the mcbR gene was deleted was identified through PCR using SEQ ID NOs: 6 and 7 for the Corynebacterium glutamicum transformant strain that was completed with secondary recombination, and this recombinant strain was named CM02-0618. I did.

CM02-0618 was deposited on January 4, 2019 with the Korean Microbiological Conservation Center, a trust organization under the Budapest Treaty, and was given the accession number KCCM12425P.

In order to analyze the L-methionine production ability of the produced CM02-0618 strain, it was cultured with the parent strain, Corynebacterium glutamicum ATCC13032, in the following manner.

Corynebacterium glutamicum ATCC13032 and Corynebacterium glutamicum CM02-0618 were inoculated into a 250 ml corner-baffle flask containing 25 ml of the seed medium below, and shaken at 30° C. for 20 hours and 200 rpm. Cultured. Then, 1 ml of the seed culture was inoculated into a 250 ml corner-baffle flask containing 24 ml of the production medium, followed by shaking culture at 30° C. for 48 hours and at 200 rpm. Compositions of the species medium and production medium are as follows, respectively. As the sulfur source in the production medium, (NH ₄ ) ₂ S ₂ O ₃ , a kind of thiosulfate, was used.

<Seed medium (pH 7.0)>

Glucose 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH ₂ PO ₄ 4 g, K ₂ HPO ₄ 8 g, MgSO ₄ 7H ₂ O 0.5 g, biotin 100 μg, thiamine HCl 1000 μg, calcium -Pantothenic acid 2000 ㎍, nicotinamide 2000 ㎍, (based on 1 liter of distilled water)

<Production medium (pH 8.0)>

Glucose 50 g, (NH ₄ ) ₂ S ₂ O ₃ 12 g, Yeast extract 5 g, KH ₂ PO ₄ 1 g, MgSO ₄ 7H ₂ O 1.2 g, biotin 100 ㎍, thiamine hydrochloride 1000 ㎍, calcium-pantothenic acid 2000 Μg, nicotinamide 3000 µg, CaCO ₃ 30 g, cyanocobalamin (Vitamin B12) 1 µg (based on 1 liter of distilled water).

After culturing by the above culture method, the L-methionine concentration in the culture solution was analyzed and shown in Table 1.

Confirmation of L-methionine production ability of wild-type and mcbR -removed strains Strain L-methionine (g/L) Corynebacterium glutamicum ATCC 13032 (wild type) 0.00 CM02-0618 0.04

As a result, it was confirmed that the L-methionine production ability was improved by 0.04g/L compared to the control strain in the mcbR- only removed strain. In addition, it was confirmed that methionine was produced even when thiosulfate was used alone as a sulfur source. Through this, it was speculated that proteins involved in the use of thiosulfate would also exist in microorganisms in the genus Corynebacterium.

Example 3: Selection of genes involved in the use of thiosulfate through transcriptome analysis

There is no known protein specific for thiosulfate of the Corynebacterium strain. However, as confirmed in Example 2, in the case of CM02-0618 strain, when thiosulfate was used as the sole sulfur source, it was confirmed that methionine was produced, and an experiment was performed to select proteins involved in the use of thiosulfate. .

Specifically, the CM02-0618 strain prepared in Example 2 was cultured with only different sulfur sources (Ammonium sulfate and ammonium thiosulfate), and then transcriptome (RNA level analysis) analysis was performed. The culture method is the same as in Example 2.

Experimental results of major gene transcripts under the conditions of ammonium sulfate and ammonium thiosulfate of CM02-0618 strain. AMS (signal) ATS (signal) Log2 ratio (ATS/AMS) SsuD(Ncgl1173) 3691 55539 2.71

As a result of the experiment, it was found that the RNA level of the gene encoding SsuD (Ncgl1173), which is known as sulfonate monooxygenase, was significantly increased.

Through this, it was confirmed that the SsuD protein did not react with sulfate but specifically reacted with thiosulfate, and it was confirmed that the protein would be involved in using thiosulfate.

Example 4: ssuD Checking the effect of gene-deficient strains

In Example 3, a vector was constructed to delete the ssuD gene in order to confirm the inactivation effect of SsuD selected as a protein that specifically reacts to thiosulfate.

Example 4-1: ssuD Vector construction for gene deletion

In order to delete the ssuD gene on the Corynebacterium ATCC13032 chromosome, a recombinant plasmid vector was constructed by the following method.

The ssuD gene and peripheral sequence (SEQ ID NO: 8) of Corynebacterium glutamicum were obtained based on the nucleotide sequence reported to the National Institutes of Health (NIH Genbank).

For the purpose of obtaining the deleted ssuD gene, PCR was performed using primers of SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12 using the chromosomal DNA of Corynebacterium glutamicum ATCC 13032 as a template. I did. PCR conditions were denatured at 95° C. for 5 minutes, then denaturation at 95° C. for 30 seconds, annealing at 53° C. for 30 seconds, and polymerization at 72° C. for 30 seconds were repeated 30 times, followed by polymerization at 72° C. for 7 minutes. As a result, each 700bp DNA fragment was obtained.

In Corynebacterium glutamicum, pDZ vector (Korean Patent Registration No. 10-0924065) and the amplified ssuD gene fragments were treated with SmaI restriction enzyme for chromosome introduction, and after isothermal assembly cloning reaction, E. coli DH5α was transformed and plated on LB solid medium containing kanamycin (25mg/ℓ). After selecting a colony transformed with a vector into which the fragments of the target genes were deleted through PCR, a plasmid was obtained using a plasmid extraction method, and was named pDZ-ΔSsuD.

Example 4-2: ssuD Creation and cultivation of strains with defective genes

The pDZ-ΔSsuD vector prepared in Example 4-1 was transformed into 13032/ΔmcbR strains respectively by electroporation by homologous recombination on chromosomes (van der Rest et al., Appl Microbiol Biotechnol 52:541- 545, 1999). Then, secondary recombination was performed in a solid medium containing sucrose. The strain in which the mcbR gene was deleted was identified through PCR using SEQ ID NOs: 13 and 14 for the Corynebacterium glutamicum transformant strain that was completed with secondary recombination, and this recombinant strain was used as Corynebacterium glue. It was named Tamicum CM02-0618/ΔSsuD.

Example 4-3: ssuD Analysis of methionine-producing ability of strains with defective genes

In order to analyze the L-methionine production ability of the produced CM02-0618/ΔSsuD strain, it was cultured with the parent strain Corynebacterium glutamicum ATCC13032 in the following manner.

In a 250 ml corner-baffle flask containing 25 ml of the following seed medium, Corynebacterium glutamicum ATCC13032 and the invention strain Corynebacterium glutamicum CM02-0618, produced CM02-0618/ΔSsuD were inoculated, and 30 Incubated with shaking at 200 rpm for 20 hours at ℃. Then, 1 ml of the seed culture was inoculated into a 250 ml corner-baffle flask containing 24 ml of the production medium, followed by shaking culture at 30° C. for 48 hours and at 200 rpm. Compositions of the species medium and production medium are as follows, respectively.

<Seed medium (pH 7.0)>

Glucose 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH ₂ PO ₄ 4 g, K ₂ HPO ₄ 8 g, MgSO ₄ 7H ₂ O 0.5 g, biotin 100 μg, thiamine HCl 1000 μg, calcium -Pantothenic acid 2000 ㎍, nicotinamide 2000 ㎍ (based on 1 liter of distilled water)

<Production medium (pH 8.0)>

Glucose 50 g, (NH ₄ ) ₂ S ₂ O ₃ 12 g, Yeast extract 5 g, KH ₂ PO ₄ 1 g, MgSO ₄ 7H ₂ O 1.2 g, biotin 100 ㎍, thiamine hydrochloride 1000 ㎍, calcium-pantothenic acid 2000 Μg, nicotinamide 3000 µg, CaCO ₃ 30 g (based on 1 liter of distilled water).

After culturing by the above culture method, the L-methionine concentration in the culture solution was analyzed and shown in Table 3.

Confirmation of L-methionine production ability of the strain from which the ssuD gene was removed Strain L-methionine (g/L) CM02-0618 0.04 CM02-0618/ΔSsuD 0.02

As a result, as the ssuD gene was removed, it was confirmed that the L-methionine production ability was reduced to about 50% level compared to the control strain. Through this, it was confirmed that the SsuD protein is a protein involved in the use of thiosulfate.

Example 5: ssuD Production and culture of strains with increased gene expression

A vector was constructed to enhance the activity of SsuD, which was selected as a protein that specifically reacts to thiosulfate in Example 3.

Example 5-1: ssuD Vector construction to increase gene expression

In order to additionally insert the ssuD gene on the Corynebacterium ATCC13032 chromosome, a recombinant plasmid vector was constructed by the following method.

First, a vector for removing Ncgl1464 (Transposase) was constructed to insert the ssuD gene.

Ncgl1464 of Corynebacterium glutamicum and surrounding sequences (SEQ ID NO: 15) were obtained based on the nucleotide sequence reported to the National Institutes of Health (NIH Genbank). In order to delete the Ncgl1464 gene, PCR was performed using primers of SEQ ID NO: 16 and SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19 using the chromosomal DNA of Corynebacterium glutamicum ATCC 13032 as a template. PCR conditions were denatured at 95° C. for 5 minutes, then denaturation at 95° C. for 30 seconds, annealing at 53° C. for 30 seconds, and polymerization at 72° C. for 30 seconds were repeated 30 times, followed by polymerization at 72° C. for 7 minutes. As a result, each DNA fragment was obtained.

In Corynebacterium glutamicum, the pDZ vector (Korean Patent Registration No. 10-0924065) and the amplified Ncgl1464 gene fragment were treated with SmaI restriction enzyme for chromosome introduction, and after isothermal assembly cloning reaction, E. coli DH5α was transformed and plated on LB solid medium containing kanamycin (25mg/ℓ). After selecting a colony transformed with a vector into which the fragments of the target genes were inserted through PCR, a plasmid was obtained using a plasmid extraction method, and it was named pDZ-ΔNcgl1464.

Next, for the purpose of obtaining the ssuD gene fragment, PCR was performed using the chromosomal DNA of Corynebacterium glutamicum ATCC 13032 as a template using SEQ ID NOs: 20 and 21. In addition, PgapA is used to enhance the expression of the ssuD gene. A promoter was used, and PCR was performed using SEQ ID NOs: 22 and 23 using Corynebacterium glutamicum ATCC 13032 chromosomal DNA as a template for the purpose of obtaining this. PCR conditions were denatured at 95° C. for 5 minutes, then denaturation at 95° C. for 30 seconds, annealing at 53° C. for 30 seconds, and polymerization at 72° C. for 30 seconds were repeated 30 times, followed by polymerization at 72° C. for 7 minutes. As a result, the ssuD gene fragment and the gapA promoter fragment were obtained.

In Corynebacterium glutamicum, pDZ-ΔNcgl1464 vector, which cannot be replicated, was treated with restriction enzyme ScaI, and then subjected to IST reaction with the two amplified DNA fragments, transformed into E. coli DH5α , and kanamycin (25mg/ It was spread on LB solid medium containing ℓ). After selecting a colony transformed with a vector into which the target gene was inserted through PCR, a plasmid was obtained using a plasmid extraction method, and was named pDZ-ΔNcgl1464-PgapASsuD.

Example 5-2: ssuD Production and culture of strains with enhanced gene expression

The pDZ-ΔNcgl1464 and pDZ-ΔNcgl1464-PgapASsuD, vectors prepared in Example 5-1 were transformed into CM02-0618 strains respectively by electroporation by homologous recombination on chromosomes (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Then, secondary recombination was performed in a solid medium containing sucrose. For the Corynebacterium glutamicum transformant, which had completed the secondary recombination, through PCR method using SEQ ID NOs: 24 and 25, the strain in which Ncgl1464 was deleted and the strain in which the ssuD gene was inserted was confirmed by the deletion of Ncgl1164. . The strain in which Ncgl1464 was deleted was named CM02-0618/ΔNcgl1464, and the strain in which the ssuD gene was inserted with the deletion of Ncgl1164 was named CM02-0736.

CM02-0736 was deposited on May 2, 2019 with the Korea Microbial Conservation Center, a trust organization under the Budapest Treaty, and was given the accession number KCCM12512P.

Example 5-3: ssuD Analysis of methionine production ability of strains with enhanced gene expression

In order to analyze the L-methionine-producing ability of the prepared CM02-0618/ΔNcgl1464 and CM02-0736 strains, they were cultured with the parent strain CM02-0618 in the following manner.

CM02-0618, CM02-0618 /ΔNcgl1464, and CM02-0736 were respectively inoculated into a 250 ml corner-baffle flask containing 25 ml of the seed medium below, and cultured with shaking at 30° C. for 20 hours and 200 rpm. Then, 1 ml of the seed culture was inoculated into a 250 ml corner-baffle flask containing 24 ml of the production medium, followed by shaking culture at 30° C. for 48 hours and at 200 rpm. Compositions of the species medium and production medium are as follows, respectively.

<Seed medium (pH 7.0)>

Glucose 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH ₂ PO ₄ 4 g, K ₂ HPO ₄ 8 g, MgSO ₄ 7H ₂ O 0.5 g, biotin 100 μg, thiamine HCl 1000 μg, calcium -Pantothenic acid 2000 ㎍, nicotinamide 2000 ㎍ (based on 1 liter of distilled water)

<Production medium (pH 8.0)>

Glucose 50 g, (NH ₄ ) ₂ S ₂ O ₃ 12 g, Yeast extract 5 g, KH ₂ PO ₄ 1 g, MgSO ₄ 7H ₂ O 1.2 g, biotin 100 ㎍, thiamine hydrochloride 1000 ㎍, calcium-pantothenic acid 2000 Μg, nicotinamide 3000 µg, CaCO ₃ 30 g, cobalamin (Vitamin B12) 1 µg (based on 1 liter of distilled water).

After culturing by the above culture method, the L-methionine concentration in the culture solution was analyzed and shown in Table 4.

Confirmation of L-methionine production ability of strains with increased expression of ssuD Strain L-methionine (g/L) CM02-0618 0.04 CM02-0618/ΔNcgl1464 0.04 CM02-0736 0.06

As a result, it was confirmed that as the ssuD gene expression was enhanced, the L-methionine production ability was increased by about 50% compared to the control strain. As confirmed in Example 4, through this, it was found that the SsuD protein is a protein involved in the use of thiosulfate.

Example 6: Comparative culture of thiosulfate and other sulfonates.

SsuD protein is originally known as a protein involved in the disulfonation of sulfonates. Sulfonate is composed of R-SO3, where R is an organic group, and thiosulfate is different from sulfonate because it is composed of S-SO3.

Thus, through a comparative experiment with sulfonate, it was attempted to confirm how much more effective the case of using thiosulfate as a sulfur source for methionine production.

Corynebacterium glutamicum CM02-0618 and CM02-0736 were inoculated into a 250 ml corner-baffle flask containing 25 ml of the seed medium below, and cultured with shaking at 30° C. for 20 hours and 200 rpm. Then, 1 ml of the seed culture solution was inoculated into a 250 ml corner-baffle flask containing 24 ml of the production medium, followed by shaking culture at 30° C. for 48 hours and 200 rpm. Compositions of the species medium and production medium are as follows, respectively.

<Seed medium (pH 7.0)>

Glucose 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH ₂ PO ₄ 4 g, K ₂ HPO ₄ 8 g, MgSO ₄ 7H ₂ O 0.5 g, biotin 100 μg, thiamine HCl 1000 μg, calcium -Pantothenic acid 2000 ㎍, nicotinamide 2000 ㎍ (based on 1 liter of distilled water)

<Production medium (pH 8.0)>

Glucose 50 g, (NH ₄ ) ₂ S ₂ O ₃ 12 g or Methanesulfonate 12 g or Ethanesulfonate 12 g (depending on sulfur sources), Yeast extract 5 g, KH ₂ PO ₄ 1 g, MgSO ₄ 7H ₂ O 1.2 g, Biotin 100 μg, thiamine hydrochloride 1000 μg, calcium-pantothenic acid 2000 μg, nicotinamide 3000 μg, CaCO ₃ 30 g, cobalamin (Vitamin B12) 1 μg (based on 1 liter of distilled water).

After culturing by the above culture method, the concentration of L-methionine in the culture solution was analyzed and shown in Table 5.

Comparison of methionine production ability with thiosulfate using various sulfonates as sulfur sources Strain Hwangwon L-methionine (g/L) CM02-0618 Thiosulfate 0.04 Metanesulfonate 0.01 ethanesulfonate 0.01 CM02-0736 Thiosulfate 0.06 Metanesulfonate 0.01 ethanesulfonate 0.02

As a result of the experiment, when thiosulfate was used as a sulfur source in each strain, methionine production was increased by up to 600% compared to when sulfonate was used as a sulfur source.

Through this, it was confirmed that methionine production was the highest when thiosulfate was used as a sulfur source, and it was confirmed that the enhancement of the activity of SsuD protein is involved in this increase in methionine production.

Example 7: mcbR Methionine-producing strain with enhanced metH and cysI expression without this defect

Example 7-1: metH, cysI A recombinant vector that simultaneously enhances

In order to enhance the SsuD protein activity of the present application and to confirm whether various sulfur-containing amino acids can be prepared when thiosulfate is used as a sulfur source, the above configuration was applied to other methionine-producing strains. Thus, in order to produce a methionine-producing strain in which mcbR is not deficient, the expression of metH (Ncgl1450) encoding a known methionine synthase and cysI (Ncgl2718) encoding a sulfite reductase is simultaneously enhanced based on the ATCC13032 strain. To do this, a vector was constructed.

Specifically, a recombinant plasmid vector was constructed by the following method in order to additionally insert the metH and cysI genes on the Corynebacterium ATCC13032 chromosome. Based on the nucleotide sequence reported to the National Institutes of Health (NIH Genbank), the metH gene and peripheral sequence (SEQ ID NO: 26), cysI gene and peripheral sequence (SEQ ID NO: 27) of Corynebacterium glutamicum were obtained. .

First, a vector for removing Ncgl1021 (Transposase) was constructed to insert them. Ncgl1021 of Corynebacterium glutamicum and surrounding sequences (SEQ ID NO: 28) were obtained based on the nucleotide sequence reported to the National Institutes of Health (NIH Genbank). For the purpose of obtaining the defective Ncgl1021 gene, PCR was performed using primers of SEQ ID NO: 29 and SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32 using the chromosomal DNA of Corynebacterium glutamicum ATCC 13032 as a template. I did. PCR conditions were denatured at 95° C. for 5 minutes, then denaturation at 95° C. for 30 seconds, annealing at 53° C. for 30 seconds, and polymerization at 72° C. for 30 seconds were repeated 30 times, followed by polymerization at 72° C. for 7 minutes. As a result, each DNA fragment was obtained. In Corynebacterium glutamicum, the pDZ vector (Korean Patent Registration No. 10-0924065) and the amplified Ncgl1021 gene fragments were treated with the restriction enzyme XbaI for chromosome introduction, and after isothermal assembly cloning reaction, E. coli DH5α was transformed and plated on LB solid medium containing kanamycin (25mg/ℓ). A plasmid was obtained using a plasmid extraction method after selecting a colony transformed with a vector into which the fragmented fragments of the target genes were inserted through PCR, and named pDZ-ΔNcgl1021.

For the purpose of obtaining the following metH and cysI genes, PCR was performed using primers of SEQ ID NO: 33 and SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36 using the chromosomal DNA of Corynebacterium glutamicum ATCC 13032 as a template. And also Pcj7 to enhance the expression of the metH gene. The Pspl1 promoter was used to enhance the expression of the promoter and cysI gene, and for the purpose of obtaining this, PCR was performed using SEQ ID NOs: 37 and 38 using Corynebacterium ammoniases ATCC 6872 chromosome DNA as a template for Pcj7. For Pspl1, PCR was performed using SEQ ID NOs: 39 and 40 using the known spl1-GFP (KR 10-1783170 B1) vector DNA as a template. PCR conditions were denatured at 95° C. for 5 minutes, then denaturation at 95° C. for 30 seconds, annealing at 53° C. for 30 seconds, and polymerization at 72° C. for 30 seconds were repeated 30 times, followed by polymerization at 72° C. for 7 minutes. As a result, DNA fragments of metH gene and cysI, Pcj7 promoter (Korean Patent No. 10-0620092) and Pspl1 promoter (Korean Patent No. 10-1783170) were obtained.

After treating the pDZ-△Ncgl1021 vector, which cannot be replicated in Corynebacterium glutamicum, with the restriction enzyme ScaI, the amplified 4 DNA fragments were treated with the restriction enzyme ScaI for chromosome introduction, and after IST reaction, E. coli Transformed into DH5α and plated on LB solid medium containing kanamycin (25mg/ℓ). After selecting a colony transformed with a vector into which the fragments of the target genes were inserted through PCR, a plasmid was obtained using a plasmid extraction method, and was named pDZ-ΔNcgl1021-Pcj7metH-Pspl1cysI.

Example 7-2: Development of L-methionine-producing strain and confirmation of L-methionine production using the same

The pDZ-ΔNcgl1021 and pDZ-ΔNcgl1021-Pcj7metH-Pspl1cysI vectors prepared in Example 7-1 were transformed into ATCC13032 strains by electroporation, respectively, by homologous recombination on chromosomes (van der Rest et al., Appl. Microbiol Biotechnol 52:541-545, 1999). Then, secondary recombination was performed in a solid medium containing sucrose. The Pcj7-metH-Pspl1cysI gene insertion was confirmed using SEQ ID NOs: 41 and 42 for the Corynebacterium glutamicum transformant strain having completed the secondary recombination. These recombinant strains were named Corynebacterium glutamicum 13032/ΔNcgl1021, CM02-0753, respectively.

In order to analyze the L-methionine production ability of the prepared 13032/ΔNcgl1021 and CM02-0753 strains, it was cultured with the parent strain, Corynebacterium glutamicum ATCC13032, in the following manner.

Corynebacterium glutamicum ATCC13032 and the invention strain Corynebacterium glutamicum 13032/ΔNcgl1021, CM02-0753 were inoculated into a 250 ml corner-baffle flask containing 25 ml of the seed medium below, and inoculated at 30° C. for 20 hours During the culture, shaking at 200 rpm. Then, 1 ml of the seed culture was inoculated into a 250 ml corner-baffle flask containing 24 ml of the production medium, followed by shaking culture at 30° C. for 48 hours and at 200 rpm. Compositions of the species medium and production medium are as follows, respectively.

<Seed medium (pH 7.0)>

Glucose 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH2PO4 4 g, K2HPO4 8 g, MgSO ₄ 7H ₂ O 0.5 g, biotin 100 μg, thiamine HCl 1000 μg, calcium-pantothenic acid 2000 μg, nicotine 2000 ㎍ of amide (based on 1 liter of distilled water)

<Production medium (pH 8.0)>

Glucose 50 g, (NH ₄ ) ₂ S ₂ O ₃ 12 g, Yeast extract 5 g, KH ₂ PO ₄ 1 g, MgSO ₄ 7H ₂ O 1.2 g, biotin 100 ㎍, thiamine hydrochloride 1000 ㎍, calcium-pantothenic acid 2000 Μg, nicotinamide 3000 µg, CaCO ₃ 30 g, cobalamin (Vitamin B12) 1 µg (based on 1 liter of distilled water).

After culturing by the above culture method, the concentration of L-methionine in the culture solution was analyzed and shown in Table 6.

Confirmation of L-methionine production ability of strains with mcbR Strain L-methionine (g/L) Corynebacterium glutamicum ATCC 13032 (wild type) 0 13032/ΔNcgl1021 0 CM02-0753 0.03

As a result, it was confirmed that mcbR was present as it was, and the strains overexpressing metH and cysI had improved L-methionine production ability compared to the control strain.

Through this, it was confirmed that mcbR was not deficient and that the strains overexpressing metH and cysI also had methionine-producing ability, and were used in the following experiments.

Example 8: mcbR L-methionine producer-based SsuD activity enhancing strain and confirmation of L-methionine production ability

Based on the methionine-producing strain prepared in Example 7, a strain having enhanced SsuD protein activity was prepared, and then L-methionine-producing ability was confirmed.

Example 8-1: SsuD enhanced strain production

Specifically, the pDZ-ΔNcgl464-PgapASsuD vector prepared in Example 5 was transformed into the CM02-0753 strain of Example 7 by electroporation by homologous recombination on a chromosome (van der Rest et al., Appl Microbiol). Biotechnol 52:541-545, 1999). Then, secondary recombination was performed in a solid medium containing sucrose.

It was confirmed whether the PgapA-SsuD gene was well inserted in the Ncgl1464 site using SEQ ID NOs: 23 and 24 for the Corynebacterium glutamicum transformant strain having completed the secondary recombination. The produced recombinant strain was named Corynebacterium glutamicum CM02-0756.

CM02-0756 was deposited with the Korean Microbiological Conservation Center, a trust organization under the Budapest Treaty, on May 2, 2019, and was given the accession number KCCM12513P.

Example 8-2: Confirmation of methionine production ability of the produced strain

In order to analyze the L-methionine-producing ability of CM02-0753 of Example 7 and CM02-0756 prepared in Example 8-1, culture was performed in the following manner.

Corynebacterium glutamicum CM02-0753 and CM02-0756 were inoculated into a 250 ml corner-baffle flask containing 25 ml of the seed medium below, and cultured with shaking at 30° C. for 20 hours at 200 rpm. Then, 1 ml of the seed culture was inoculated into a 250 ml corner-baffle flask containing 24 ml of the production medium, followed by shaking culture at 30° C. for 48 hours and at 200 rpm. Compositions of the species medium and production medium are as follows, respectively.

<Seed medium (pH 7.0)>

Glucose 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH ₂ PO ₄ 4 g, K ₂ HPO ₄ 8 g, MgSO ₄ 7H ₂ O 0.5 g, biotin 100 μg, thiamine HCl 1000 μg, calcium -Pantothenic acid 2000 ㎍, nicotinamide 2000 ㎍ (based on 1 liter of distilled water)

<Production medium (pH 8.0)>

Glucose 50 g, (NH ₄ ) ₂ S ₂ O ₃ 12 g, Yeast extract 5 g, KH ₂ PO ₄ 1 g, MgSO ₄ 7H ₂ O 1.2 g, biotin 100 ㎍, thiamine hydrochloride 1000 ㎍, calcium-pantothenic acid 2000 Μg, nicotinamide 3000 µg, CaCO ₃ 30 g, cobalamin (Vitamin B12) 1 µg (based on 1 liter of distilled water).

After culturing by the above culture method, the concentration of L-methionine in the culture solution was analyzed and shown in Table 7.

Confirmation of L-methionine production ability when ssuD gene is overexpressed in strains with mcbR Strain L-methionine (g/L) CM02-0753 0.03 CM02-0756 0.05

As a result, it was confirmed that when the SsuD activity was enhanced in the methionine strain in which mcbR was present, when thiosulfate was used as a sulfur source, the methionine yield increased.

These results suggest that when SsuD activity, a protein involved in the use of thiosulfate, which has been newly identified in the present application, is enhanced, a sulfur-containing amino acid or a sulfur-containing amino acid derivative can be prepared based on thiosulfate, a sulfur source. will be.

From the above description, those skilled in the art to which the present application belongs will understand that the present application may be implemented in other specific forms without changing the technical spirit or essential features. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present application should be construed as including all changes or modified forms derived from the meaning and scope of the claims to be described later rather than the above detailed description, and equivalent concepts thereof.

Korea Microorganism Conservation Center (overseas) KCCM12425P 20190104 Korea Microorganism Conservation Center (overseas) KCCM12512P 20190502 Korea Microorganism Conservation Center (overseas) KCCM12513P 20190502

<110> CJ CheilJedang Corporation <120> A method of producing sulfur-containing amino acids and derivatives thereof <130> KPA190476-KR <160> 43 <170> KoPatentIn 3.0 <210> 1 <211> 2642 <212> DNA <213> Corynebacterium glutamicum <400> 1 ctcccgcgca ctgctgcaat ccgcaccgtg cccaatgatg gtggttcgcc cacctgagaa 60 gattaagaag tagtttcttt taagtttcga tgccccggtt tcctgatttt gtgcagggag 120 gccggggcat tggtgtttgc gggttagttc gggccattcg aaagggagaa accaagggca 180 gccagacaga cgtgccaaga atctggattt ccgccaggtt ttggcacgcc cgtctggttt 240 aggcaatgag ataccgaaca cacgtgccaa aagttcggct ttttcgccga tcttgtcacg 300 cctgcctggt ttgtcttgta aagagtgatt tcatggccga gactcctaaa agtttgacct 360 cacaggattg cttctaaggg cctctccaat ctccactgag gtacttaatc cttccgggga 420 attcgggcgc ttaaatcgag aaattaggcc atcacctttt aataacaata caatgaataa 480 ttggaatagg tcgacacctt tggagcggag ccggttaaaa ttggcagcat tcaccgaaag 540 aaaaggagaa ccacatgctt gccctaggtt ggattacatg gatcattatt ggtggtctag 600 ctggttggat tgcctccaag attaaaggca ctgatgctca gcaaggaatt ttgctgaaca 660 tagtcgtcgg tattatcggt ggtttgttag gcggctggct gcttggaatc ttcggagtgg 720 atgttgccgg tggcggcttg atcttcagct tcatcacatg tctgattggt gctgtcattt 780 tgctgacgat cgtgcagttc ttcactcgga agaagtaatc tgctttaaat ccgtagggcc 840 tgttgatatt tcgatatcaa caggcctttt ggtcattttg gggtggaaaa agcgctagac 900 ttgcctgtgg attaaaacta tacgaaccgg tttgtctata ttggtgttag acagttcgtc 960 gtatcttgaa acagaccaac ccgaaaggac gtggccgaac gtggctgcta gcgcttcagg 1020 caagagtaaa acaagtgccg gggcaaaccg tcgtcgcaat cgaccaagcc cccgacagcg 1080 tctcctcgat agcgcaacca accttttcac cacagaaggt attcgcgtca tcggtattga 1140 tcgtatcctc cgtgaagctg acgtggcgaa ggcgagcctc tattcccttt tcggatcgaa 1200 ggacgccttg gttattgcat acctggagaa cctcgatcag ctgtggcgtg aagcgtggcg 1260 tgagcgcacc gtcggtatga aggatccgga agataaaatc atcgcgttct ttgatcagtg 1320 cattgaggaa gaaccagaaa aagatttccg cggctcgcac tttcagaatg cggctagtga 1380 gtaccctcgc cccgaaactg atagcgaaaa gggcattgtt gcagcagtgt tagagcaccg 1440 cgagtggtgt cataagactc tgactgattt gctcactgag aagaacggct acccaggcac 1500 cacccaggcg aatcagctgt tggtgttcct tgatggtgga cttgctggat ctcgattggt 1560 ccacaacatc agtcctcttg agacggctcg cgatttggct cggcagttgt tgtcggctcc 1620 acctgcggac tactcaattt agtttcttca ttttccgaag gggtatcttc gttgggggag 1680 gcgtcgataa gccccttctt tttagcttta acctcagcgc gacgctgctt taagcgctgc 1740 atggcggcgc ggttcatttc acgttgcgtt tcgcgcctct tgttcgcgat ttctttgcgg 1800 gcctgttttg cttcgttgat ttcggcagta cgggttttgg tgagttccac gtttgttgcg 1860 tgaagcgttg aggcgttcca tggggtgaga atcatcaggg cgcggttttt gcgtcgtgtc 1920 cacaggaaga tgcgcttttc tttttgtttt gcgcggtaga tgtcgcgctg ctctaggtgg 1980 tgcactttga aatcgtcggt aagtgggtat ttgcgttcca aaatgaccat catgatgatt 2040 gtttggagga gcgtccacag gttgttgctg acccaataga gtgcgattgc tgtggggaat 2100 ggtcctgtga ggccaaggga cagtgggaag atcggcgcga ggatcgacat cacgatcatg 2160 aacttcagca tgccgttaga gaatccggat gcgtaatcgt tggtttggaa gctgcggtac 2220 atggacatcg ccatgttgat tgcggtgagg attgcggctg tgatgaacag tggcaaaacg 2280 aaactaagaa cttccgcctg cgtggtgctc aaatatttta gctgctcagt gggcatcgaa 2340 acataagcgg gcagaggcac attgctcacg cgaccagcga ggaaagattc cacttcctca 2400 ggagttagga agccgatcga ctggaagacg ggattttcca aaccaccttc agggcgagcc 2460 atgcggagaa gtgcccagta aagaccaagg acaatcggta tctggatcag cccaggcaca 2520 caacctgcca gcgggttaat gccgtattcc ttattcaaat cattctggcg cttctgcaac 2580 tcccgaatgg acgcttcatc gtactttccc ttgtattctt cccggagcgc agcgcggtga 2640 gg 2642 <210> 2 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 tcgagctcgg tacccctgcc tggtttgtct tgta 34 <210> 3 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 cggaaaatga agaaagttcg gccacgtcct ttcgg 35 <210> 4 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 aggacgtggc cgaactttct tcattttccg aaggg 35 <210> 5 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 ctctagagga tccccgtttc gatgcccact gagca 35 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 aatctggatt tccgccaggt 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 cttcctaact cctgaggaag 20 <210> 8 <211> 3146 <212> DNA <213> Corynebacterium glutamicum <400> 8 ccatggagaa cctggagaat gaggtgctgc gtcgttccac gcaggttccg gtgattgttc 60 tcgtgggtac cccgcgcagc cctgattcgg agcagttgaa gtcggatctg accacgcttg 120 ctgctgaaag tggcaggaag ttcattttcg gttatgtcaa tgctgatacc gatgctgatg 180 tggcccaggt gtttggggtg cagggcttgc cgtcggtgat tgctgtggca gcgggacgcc 240 ctctggctga tttccagggc ggacagccag cggatgcact aaagcagtgg actgatcagg 300 tggttcaggc tgtgggtgga cagctggaag gactgccaga ggaggccaca gacggcgaac 360 aagaagacgc tcctgtggaa gacccccgct tcgatgctgc cactgatgct ctaaaccgtg 420 gcgctttcga tgaggcgatt gcggtttatg agtccatttt ggcgcaggag ccaaacaacg 480 ctgatgcgaa gcaggcacgc gataccgcaa agctgttggg ccggcttgcc acggtggatc 540 cttcggtgga tgttgtcgct gctgcagatg ctgatccaac aaacgttgat ctggcctaca 600 cagcagctga cgcggctgtt gttgcgggtg atcctgaggc tgcctttgat cgtttaattg 660 ctctgctgac catcagcgct ggcgatcaga agaatcaggt gaaggaacgt ttgctggagc 720 tgtttggcat gtttgagacc gccgatcccc gtgtgctgca ggcgcgagga aagatggcca 780 gcgcgctgtt ctaaaaccac tctctatcca gaaaaatata gaccgcttag tcttttccag 840 gactaagtgg tctacatttt tacccaaaat gcagctcacg caatagacat ctcggtctat 900 atacttgcca ccttcgcgcc ctgcaaatcc ccacactact tatatccagc ccgaaaataa 960 tacttctctc tagacgaagc ggtctgttta agtatgtgcc atgacattaa ctttccattg 1020 gttcctatcc acttcaggcg attcccgcgg catcatcggc ggcggtcacg gtgcagaaaa 1080 atccggcacc tcccgcgaat tgagccacag ctacctcaag cagttggcgc tagctgccga 1140 gaccaacggt tttgaatctg tcctgacacc aacgggcacg tggtgcgaag atgcgtggat 1200 tactgacgct tctttgattg aggcgacaaa acgcttgaag ttcctcgttg cgcttcgccc 1260 tgggcagatt ggacctacgc tgtctgctca aatggcttct actttccagc gtctgtctgg 1320 caaccgtttg ctgatcaatg tggtcaccgg tggggaagat gcggagcagc gtgcgtttgg 1380 tgatttcttg aacaaggagg agcgctacgc ccgtaccgga gaattcttgg atatcgtgag 1440 ccgcttgtgg cgaggcgaaa ccgtcacgca ccacggtgaa cacctgcagg tggagcaagc 1500 tagccttgcg catccgccag agattattcc ggagattctt tttggtggat cgtcgccagc 1560 tgcaggtgag gtggctgcac gttatgcgga cacctatctc acgtggggtg aaactcccga 1620 tcaggtggcg cagaaaatca actggatcaa cgagctagca gcacagcgcg gccgggaact 1680 gcgccatgga atccgcttcc atgtgatcac ccgcgatacg tctgaagaag catgggtggt 1740 ggcagagaag ttgattagcg gggtcactcc agaacaggtc gctaaggctc aagccgggtt 1800 tgcaacgtct aagtcggagg ggcagcgccg gatggctgag ctgcacagca agggtcgtgc 1860 ctttactagt ggctcaactg ctcgtgatct ggaggtgtat cccaatgtgt gggcaggcgt 1920 cggtttgctt cgcggaggtg caggaacagc ccttgtgggc tcgcatgaag aggtcgccga 1980 tcgcatcgaa gaatacgcag cactcggctt ggatcagttt gtactgtcgg gttatccaaa 2040 cttggaggag gccttccact tcggtgaggg tgtgattccg gagctgctgc gccgcggtgt 2100 ggatatcaaa aatcaagaat cacgagtttt ggaacctgtt gggtaaacgg gaagaacgag 2160 acgtcgataa gcaaatttct taaggaacct gacatgacta caaccttgac tcgccccaaa 2220 atcgcgctgc ccgcgcgcat ctattcaccg cttgcggtgc ttgttttctg gcagctcggc 2280 tcgagcctgg gcgccatccc ggagcggatt ctgccggcac caaccacgat cttggccgcc 2340 agctgggagg tcgccacaaa tggcacgctt ctcgacgccc tcctcgtctc aagccaacgc 2400 gtccttctag gcttcgccct cggtgctgtc ctaggcattt ccctaggtgt attgacaggc 2460 atgtccagat ttgcagacac cgccgttgat ccgctcattc aagctgcccg cgcgctgcct 2520 cacctgggtc ttgtgccgct gtttatcatc tggttcggta tcggtgagct gccgaaagta 2580 ctgattatta gcctcggcgt gctgtatccg ctgtacctca acaccgccag cgggttcagg 2640 caaattgatc caaagcttct ggaagccggc cacgtgatgg gcttcggatt tttccagagg 2700 ttgcggacca tcatcattcc ttctgccgcg ccgcaacttt ttgtcggcct gcgccaagca 2760 agtgcggccg cctggctctc actgatcgtg gcggaacagg tcaacgcccg cgaaggactc 2820 ggcttcctca tcaacaatgc gcgcgatttt taccgcaccg acctcgttat tttcggcctc 2880 attgtctacg ccagcctcgg tctgctgtct gaagcgctga tcagagcttg ggaacgtcac 2940 accttccgct accgaaacgc ataagaaagt tgctcgccat gactgccaca ttgtcactca 3000 aacccgcagc cactgtccgt ggattgcgca aatcatacgg aactaaagaa gtcctccaag 3060 gaatcgacct caccatcaac tgcggcgaag taaccgcgct gatcggacgc tcaggttcag 3120 gaaaatccac catcctgcgc gtgttg 3146 <210> 9 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 tcgagctcgg taccctggtt caggctgtgg gtgga 35 <210> 10 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 tcttcccgta gtactggcac atacttaaac agacc 35 <210> 11 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 gtatgtgcca gtactacggg aagaacgaga cgtcg 35 <210> 12 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 ctctagagga tcccctcgcg cgcattgttg atgag 35 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 ccaggtgttt ggggtgcagg 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 aatccacgga cagtggctgc 20 <210> 15 <211> 3161 <212> DNA <213> Corynebacterium glutamicum <400> 15 agacgattct ggaaggccac tttcttttga tcctggcggc gatttttcgg tgcttggtag 60 cggaatgtca atcgagacca agcgtccccc ggcgggtcga ttttttggtc tcgaatgaca 120 gtttcgctct ctggaaactc tcagtgtcag gtcagtggtg aaccaccatc cttagcaagg 180 agttcatcat gtccatcccc ttctcagtcc ttcaggacta cctggatctg atcagtcccg 240 aagccttacc ccagatccca cagcccccgg cccctgcccc cacagcaccc cagctaccac 300 cggcgccgga cccacacagc atcgagtggc cgatcttccc accagatcga atctccgcca 360 acgggcgacg ctactacgag ccacaaacac gactcgagtt catgcggatc tacaccaccc 420 tgccgcacgg ctaccgccag cccttcctta aagccaacaa catcggccac tgcaccgttc 480 gaacctggct agcagcaata agcaccttca gccgacttcc ccatgctttt gatgatgccc 540 accgcttcgg gatcgaacgc accaccccag tcgacgatgt caccacacta acggctgatg 600 acaaacgtga cctggtcata ggatacttag ctcaaccaca cggtcagggc cagcaattcc 660 tcacgtttta ccaactccgt aagcacacca tcatggcctg gtgcgccgct atgaccgacg 720 gggacttaga cgctgatatc tcaccccgcc agatcgggtt gatgaccacc cgaaccgtgg 780 tcgaaatcgt tcgactacgc cacatgattg cccaacaact agaaagagcc acgatcatgg 840 aaaacgagta cctcaaagaa atcgcagcgc tgaagaaaga actcgcgcac tacaagcaaa 900 aagaccatca gaatcaaatg gtgatcgata tcttgggaaa agctattggg accaggccca 960 atcctggcga gggcttagac gaggaggacg ccacctaaac gtggatgagc aacgcgcctt 1020 tgatcaagga ctcaaggaag aaaacacctt gatcacagat ctcaccacct gtgccaggct 1080 gagccataac aaggcattac ggctgatcaa gctgtcgaaa tcaacggcgt attaccgcaa 1140 caagccgcgt ccccgtcctg caccgaaacc tgtcctgcag gccgtgccag caccaacagc 1200 acctggtgtg gaacccacac cagagccttg gcaggggaag gagccagcag tgtcgtcggt 1260 gcgtcaagcg ttggcagaac acgaacgcca gttcattgtt gatgcgatca ccgcgtaccc 1320 acaactgagc gttagtgggg tgtttaacat gttgtttaac aaaggcatct accgcgcatc 1380 actacgtaca tggtggcgtg ttgccaagca gcacaagttg ttacacaaag accgagtcag 1440 tgccctgtcc ccggggaaac gatcaccaac gccacgggtt aagccgaggt tggaagcaac 1500 acagcctggt caggtggtgt gttgggatgt gacgttcttg ccgtcgctgg tacgtggtaa 1560 gacctatgcg ttgcatctgg cgattgattt gttttcccgc aagattgttg gggcgaaggt 1620 cgcgccgacg gaaaatacct ccaccgcggt ggagttgtta acgcaggtgt tagcggataa 1680 tccgggtgtg gtgacggtgc attcggataa tgggtcggcg atgacatcga cgagggtgcg 1740 gcggttgtta gcggatcatg gtgtggcgtt gtcgttgatt cggccgcggg tgagtgatga 1800 taatgcgttt gtggagtcgg tgtttcatac gttgaagtat cggccgtttt atccgaaggt 1860 gtttgcatcg atggatcagg cccgggtgtg ggtggaggag tttgtggtgt attacaacac 1920 ggttcatccg cattctggtg tggctgggca tactccgcag tcggtgtttg atggtagttg 1980 gagggcggct cataggttgc gtgtgcaggc gttggatgcc cattaccggc agttcccgca 2040 gcggtatgtg gggcggccgg tggttcagga agttgctggt gtggtgcgtc ttaatggtgc 2100 gcgtgatgat gggtctgtac aggagagggt tggtggtgta gcgtcgctgt taagtgcttg 2160 agttagcatg tgttcttatc gcccccctgg ttcacaaacc cctggcagcg agcggaaaag 2220 tgcattttta ggccaagggc cctcggatct tcgagcgctt tggtctcttt tgcacgtctg 2280 accgaaccag atcacctaga aacgccaaag gccccgcaag tatcaaacct gcggggcctt 2340 tgaggtacct gtttcctatt ttgttgactt aggaagctgc gcacggcgga taaccaaacc 2400 gcacagcaag gcagccactc cccacgcggt gagccagaac tgctccacga cataaacctg 2460 aatagttgga agcaaacgac ttacgatcac cagggctaca gcgatgctca aagaaatgat 2520 ctgtgtcttt gagcttggct cgtacttgct ggtggtagcg aatgcgccga gaatgatcga 2580 ggcaacgagg atcagaagga atcctggagt gatcacaaat gggctcagca tgccggcggt 2640 aaacagctca attgctgcaa acaccaacaa aaccagacct aaggctacga aagctccacc 2700 gatgcggatt gctgccggaa ctttacgcca gctggcacgg ccttcgaggc tggtgagctt 2760 ttttaatgat cttcccgatg ctgaactcat aatgtgacat accctactag ttctcgtacc 2820 atccccacac aattgacctg ccaagagtgt ggaaatacag gttgaagcct agaacagtgg 2880 gggtagcgtc gggggcgatg tcgagttttt ccacatcaag tgcatgaact gcgaagaggt 2940 aacggtgcgg tgcgtggcca gctggaggtt gcgctccgta gaagccacgc ttgccggaat 3000 cacccttgag ggaaactacg ccttcgatgc cgccgagggt ttcatcgcca gcaccggtgg 3060 ggatctccgt gacagttgtg gggatgttaa acactgccca gtgccagaaa ccagcgccgg 3120 ttggggcatc tgggtcgagg caggtgatcg cgagggattt g 3161 <210> 16 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 tcgagctcgg tacccttttt tggtctcgaa tgaca 35 <210> 17 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 catgctaaca gtactgttta ggtggcgtcc tcctc 35 <210> 18 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 gtatgtgcca gtactacggg aagaacgaga cgtcg 35 <210> 19 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 ctctagagga tcccctcgcg cgcattgttg atgag 35 <210> 20 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 tagaggagac acaacatgac attaactttc cattg 35 <210> 21 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 acacatgcta acagtttacc caacaggttc caaaa 35 <210> 22 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 cgccacctaa acagtgaagc ctaaaaacga ccgag 35 <210> 23 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 gaaagttaat gtcatgttgt gtctcctcta aagat 35 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 24 ctggcggcga tttttcggtg 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 25 cgcgatcacc tgcctcgacc 20 <210> 26 <211> 5666 <212> DNA <213> Corynebacterium glutamicum <400> 26 tgtcatgctt ccggaggtgc gcagggctcg agactccgga aagctatttg ccactccgat 60 gtttgggtca ctcgacgaga tacgtgctga tcacctaatt tggtgcacag ggtttcggcc 120 ggcgattagg ccagttcgtc aacttctcaa acacggacaa ccaaaggttc ctggtcttta 180 tttagtaggc tacggagatt ggacgggacc tgggtctgcg actatcacag gggtcgggct 240 ttatgccaag cgagcagcca aagagattgc cgcgtcagtc ggcaaagtcg ttaaatagtt 300 tgaaggctaa gaacttaatg ttaaagcgaa aattgttttg acacctcaac taatgcagcg 360 atgcgttctt tccagaatgc tttcatgaca gggatgctgt cttgatcagg caggcgtctg 420 tgctggatgc cgaagctgga tttattgtcg cctttggagg tgaagttgac gctcactcga 480 gaatcatcgg ccaaccattt ggcattgaat gttctaggtt cggaggcgga ggttttctca 540 attagtgcgg gatcgagcca ctgcgcccgc aggtcatcgt ctccgaagag cttccacact 600 ttttcgaccg gcaggttaag ggttttggag gcattggccg cgaacccatc gctggtcatc 660 ccgggtttgc gcatgccacg ttcgtattca taaccaatcg cgatgccttg agcccaccag 720 ccactgacat caaagttgtc cacgatgtgc tttgcgatgt gggtgtgagt ccaagaggtg 780 gcttttacgt cgtcaagcaa ttttagccac tcttcccacg gctttccggt gccgttgagg 840 atagcttcag gggacatgcc tggtgttgag ccttgcggag tggagtcagt catgcgaccg 900 agactagtgg cgctttgcct gtgttgctta ggcggcgttg aaaatgaact acgaatgaaa 960 agttcgggaa ttgtctaatc cgtactaagc tgtctacaca atgtctactt cagttacttc 1020 accagcccac aacaacgcac attcctccga atttttggat gcgttggcaa accatgtgtt 1080 gatcggcgac ggcgccatgg gcacccagct ccaaggcttt gacctggacg tggaaaagga 1140 tttccttgat ctggaggggt gtaatgagat tctcaacgac acccgccctg atgtgttgag 1200 gcagattcac cgcgcctact ttgaggcggg agctgacttg gttgagacca atacttttgg 1260 ttgcaacctg ccgaacttgg cggattatga catcgctgat cgttgccgtg agcttgccta 1320 caagggcact gcagtggcta gggaagtggc tgatgagatg gggccgggcc gaaacggcat 1380 gcggcgtttc gtggttggtt ccctgggacc tggaacgaag cttccatcgc tgggccatgc 1440 accgtatgca gatttgcgtg ggcactacaa ggaagcagcg cttggcatca tcgacggtgg 1500 tggcgatgcc tttttgattg agactgctca ggacttgctt caggtcaagg ctgcggttca 1560 cggcgttcaa gatgccatgg ctgaacttga tacattcttg cccattattt gccacgtcac 1620 cgtagagacc accggcacca tgctcatggg ttctgagatc ggtgccgcgt tgacagcgct 1680 gcagccactg ggtatcgaca tgattggtct gaactgcgcc accggcccag atgagatgag 1740 cgagcacctg cgttacctgt ccaagcacgc cgatattcct gtgtcggtga tgcctaacgc 1800 aggtcttcct gtcctgggta aaaacggtgc agaataccca cttgaggctg aggatttggc 1860 gcaggcgctg gctggattcg tctccgaata tggcctgtcc atggtgggtg gttgttgtgg 1920 caccacacct gagcacatcc gtgcggtccg cgatgcggtg gttggtgttc cagagcagga 1980 aacctccaca ctgaccaaga tccctgcagg ccctgttgag caggcctccc gcgaggtgga 2040 gaaagaggac tccgtcgcgt cgctgtacac ctcggtgcca ttgtcccagg aaaccggcat 2100 ttccatgatc ggtgagcgca ccaactccaa cggttccaag gcattccgtg aggcaatgct 2160 gtctggcgat tgggaaaagt gtgtggatat tgccaagcag caaacccgcg atggtgcaca 2220 catgctggat ctttgtgtgg attacgtggg acgagacggc accgccgata tggcgacctt 2280 ggcagcactt cttgctacca gctccacttt gccaatcatg attgactcca ccgagccaga 2340 ggttattcgc acaggccttg agcacttggg tggacgaagc atcgttaact ccgtcaactt 2400 tgaagacggc gatggccctg agtcccgcta ccagcgcatc atgaaactgg taaagcagca 2460 cggtgcggcc gtggttgcgc tgaccattga tgaggaaggc caggcacgta ccgctgagca 2520 caaggtgcgc attgctaaac gactgattga cgatatcacc ggcagctacg gcctggatat 2580 caaagacatc gttgtggact gcctgacctt cccgatctct actggccagg aagaaaccag 2640 gcgagatggc attgaaacca tcgaagccat ccgcgagctg aagaagctct acccagaaat 2700 ccacaccacc ctgggtctgt ccaatatttc cttcggcctg aaccctgctg cacgccaggt 2760 tcttaactct gtgttcctca atgagtgcat tgaggctggt ctggactctg cgattgcgca 2820 cagctccaag attttgccga tgaaccgcat tgatgatcgc cagcgcgaag tggcgttgga 2880 tatggtctat gatcgccgca ccgaggatta cgatccgctg caggaattca tgcagctgtt 2940 tgagggcgtt tctgctgccg atgccaagga tgctcgcgct gaacagctgg ccgctatgcc 3000 tttgtttgag cgtttggcac agcgcatcat cgacggcgat aagaatggcc ttgaggatga 3060 tctggaagca ggcatgaagg agaagtctcc tattgcgatc atcaacgagg accttctcaa 3120 cggcatgaag accgtgggtg agctgtttgg ttccggacag atgcagctgc cattcgtgct 3180 gcaatcggca gaaaccatga aaactgcggt ggcctatttg gaaccgttca tggaagagga 3240 agcagaagct accggatctg cgcaggcaga gggcaagggc aaaatcgtcg tggccaccgt 3300 caagggtgac gtgcacgata tcggcaagaa cttggtggac atcattttgt ccaacaacgg 3360 ttacgacgtg gtgaacttgg gcatcaagca gccactgtcc gccatgttgg aagcagcgga 3420 agaacacaaa gcagacgtca tcggcatgtc gggacttctt gtgaagtcca ccgtggtgat 3480 gaaggaaaac cttgaggaga tgaacaacgc cggcgcatcc aattacccag tcattttggg 3540 tggcgctgcg ctgacgcgta cctacgtgga aaacgatctc aacgaggtgt acaccggtga 3600 ggtgtactac gcccgtgatg ctttcgaggg cctgcgcctg atggatgagg tgatggcaga 3660 aaagcgtggt gaaggacttg atcccaactc accagaagct attgagcagg cgaagaagaa 3720 ggcggaacgt aaggctcgta atgagcgttc ccgcaagatt gccgcggagc gtaaagctaa 3780 tgcggctccc gtgattgttc cggagcgttc tgatgtctcc accgatactc caaccgcggc 3840 accaccgttc tggggaaccc gcattgtcaa gggtctgccc ttggcggagt tcttgggcaa 3900 ccttgatgag cgcgccttgt tcatggggca gtggggtctg aaatccaccc gcggcaacga 3960 gggtccaagc tatgaggatt tggtggaaac tgaaggccga ccacgcctgc gctactggct 4020 ggatcgcctg aagtctgagg gcattttgga ccacgtggcc ttggtgtatg gctacttccc 4080 agcggtcgcg gaaggcgatg acgtggtgat cttggaatcc ccggatccac acgcagccga 4140 acgcatgcgc tttagcttcc cacgccagca gcgcggcagg ttcttgtgca tcgcggattt 4200 cattcgccca cgcgagcaag ctgtcaagga cggccaagtg gacgtcatgc cattccagct 4260 ggtcaccatg ggtaatccta ttgctgattt cgccaacgag ttgttcgcag ccaatgaata 4320 ccgcgagtac ttggaagttc acggcatcgg cgtgcagctc accgaagcat tggccgagta 4380 ctggcactcc cgagtgcgca gcgaactcaa gctgaacgac ggtggatctg tcgctgattt 4440 tgatccagaa gacaagacca agttcttcga cctggattac cgcggcgccc gcttctcctt 4500 tggttacggt tcttgccctg atctggaaga ccgcgcaaag ctggtggaat tgctcgagcc 4560 aggccgtatc ggcgtggagt tgtccgagga actccagctg cacccagagc agtccacaga 4620 cgcgtttgtg ctctaccacc cagaggcaaa gtactttaac gtctaacacc tttgagaggg 4680 aaaactttcc cgcacattgc agatcgtgcc actttaacta aggttgacgg catgattaag 4740 gcgattttct gggacatgga cggcacgatg gtggactctg agccacagtg gggcattgct 4800 acctacgagc tcagcgaagc catgggccgc cgcctcaccc cggagctccg ggaactcacc 4860 gtcggctcga gcctgccgcg caccatgcgc ttatgcgcag agcacgcagg cattacattg 4920 agcgacgcgg actacgagcg ctaccgggct ggcatgttcg cccgggtcca tgagcttttc 4980 gacgaatccc tcgtcccaaa tccaggcgtc accgaactcc tgacagagtt gaaggccctc 5040 gagatcccca tgttggtcac caccaacaca gagcgcgatc tcgcgacccg ttcagtcgca 5100 gccgtgggaa atgagttctt catcggttct atcgctggtg atgaagtccc aacagcaaag 5160 ccagcccccg acatgtacct cgaagcagca cgacgtgtgg gctttgaccc atcagagtgc 5220 ctcgtgttcg aagattccta caacggcatg ctgggcgctg ttactgcagg ttgccgcgtc 5280 attggtctgc acccagaaga agtccaagcg ccagaaggtg tagtgccttt gcgttccctc 5340 cacggtaaaa actctttcga aggtgtcacc gctgagatgg tcactgcctg gtaccaccag 5400 atcgagccgg caggtgtcgc aaaataaaac caggtggggg agtgaaatta ttcgactaat 5460 atcctccccc aaacacacat tgataactgt tgtgtggaag aatgtaccga gtgaagacat 5520 ttgactcgct gtacgaagaa cttcttaacc gtgctcagac ccgccctgaa gggtctggaa 5580 ccgtggccgc cttggataaa ggcatccatc atctaggtaa gaaggtcatc gaagaagccg 5640 gagaggtctg gattgcagcc gagtat 5666 <210> 27 <211> 3613 <212> DNA <213> Corynebacterium glutamicum <400> 27 tcctgtgggg tgaacttgac ctgtgctggg ccacgacgtc cgaaaacgtg cacttcagtg 60 gccttgtttt ctttgaggga gtcgtagacg ttgtcggaaa tttcggtgac tttgagctcg 120 tcgcctgtct tagccaggat gcgggctacg tcgaggccga cgttaccaac gccgataaca 180 gcgacggact gtgcagacag atcccaggag cgctcgaagc gtgggttgcc gtcgtagaag 240 ccaacgaact cgccggcacc gaaggagcct tctgcttcaa ttccggggat gttgaggtcg 300 cggtctgcaa ctgcgccggt ggagaacacg actgcatcgt agtagtcgcg gagttcttcg 360 acggtgatgt ctttgccgat ttcaatgtta ccgagcaggc gcaggcgtgg cttgtccaac 420 acgttgtgca gggacttaac gatgcccttg atgcgtgggt ggtctggagc aacgccgtaa 480 cggatgagtc cgaacggtgc aggcatttgc tcgaaaaggt caacgaacac ttcgcgctct 540 tcattgcgga tgaggaggtc ggatgcgtaa atgccagcag ggccagctcc gatgacggct 600 acgcgcaggg gagttgtcat gtgtttgaag ttgcctttcg tgagcccttt tatggaaaca 660 agggtgtgaa aatcaagtag ttaaaggtgt ttcaagtcca ggctgtttaa cactcctaga 720 ccgcttggtc tgtaaacgta gcagcgaaat gcgacaatgc gaagactttt gcttaattaa 780 attcaaactc catgaaaaaa ctagacagat cggtctatta tattcacggt gaacctaacc 840 taatatcccc aggttaattc atttaaacgg gcattaggtg actccattgc tttcagtctc 900 atgaatctaa tggttggtct agacagagcg gtacgtctaa gtttgcggat agatcaaacc 960 gagtgacatg tacttcacta gctctttaag gattaactcc ccatgacaac aaccaccgga 1020 agtgcccggc cagcacgtgc cgccaggaag cctaagcccg aaggccaatg gaaaatcgac 1080 ggcaccgagc cgcttaacca tgccgaggaa attaagcaag aagaacccgc ttttgctgtc 1140 aagcagcggg tcattgatat ttactccaag cagggttttt cttccattgc accggatgac 1200 attgccccac gctttaagtg gttgggcatt tacacccagc gtaagcagga tctgggcggt 1260 gaactgaccg gtcagcttcc tgatgatgag ctgcaggatg agtacttcat gatgcgtgtg 1320 cgttttgatg gcggactggc ttcccctgag cgcctgcgtg ccgtgggtga aatttctagg 1380 gattatgctc gttccaccgc ggacttcacc gaccgccaga acattcagct gcactggatt 1440 cgtattgaag atgtgcctgc gatctgggag aagctagaaa ccgtcggact gtccaccatg 1500 cttggttgcg gtgacgttcc acgtgttatc ttgggctccc cagtttctgg cgtagctgct 1560 gaagagctga tcgatgccac cccggctatc gatgcgattc gtgagcgcta cctagacaag 1620 gaagagttcc acaaccttcc tcgtaagttt aagactgcta tcactggcaa ccagcgccag 1680 gatgttaccc acgaaatcca ggacgtttcc ttcgttcctt cgattcaccc agaattcggc 1740 ccaggatttg agtgctttgt gggcggtggc ctgtccacca acccaatgct tgctcagcca 1800 cttggttctt ggattccact tgatgaggtt ccagaagtgt gggctggcgt cgccggaatt 1860 ttccgcgact acggcttccg acgcctgcgt aaccgtgctc gcctcaagtt cttggtggca 1920 cagtggggta ttgagaagtt ccgtgaagtt cttgagaccg aatacctcga gcgcaagctg 1980 atcgatggcc cagttgttac caccaaccct ggctaccgtg accacattgg cattcaccca 2040 caaaaggacg gcaagttcta cctcggtgtg aagccaaccg ttggacacac caccggtgag 2100 cagctcattg ccattgctga tgttgcagaa aagcacggca tcaccaggat tcgtaccacg 2160 gcggaaaagg aactgctctt cctcgatatt gagagaaaga accttactac cgttgcacgc 2220 gacctggatg aaatcggact gtactcttca ccttccgagt tccgccgcgg catcatttcc 2280 tgcaccggct tggagttctg caagcttgcg cacgcaacca ccaagtcacg agcaattgag 2340 cttgtcgacg aactggaaga gcgcctcggc gatttggatg ttcccatcaa gattgcactg 2400 aacggttgcc ctaactcttg tgcacgcacc caggtttccg acatcggatt caagggacag 2460 accgtcactg atgctgacgg caaccgcgtt gaaggtttcc aggttcacct gggcggttcc 2520 atgaacttgg atccaaactt cggacgcaag ctcaagggcc acaaggttat tgccgatgaa 2580 gtgggagagt acgtcactcg cgttgttacc cacttcaagg aacagcgcca cgaggacgag 2640 cacttccgcg attgggtcca gcgggccgct gaggaagatt tggtgtgagt cttcggagga 2700 aacccaatcc caaccgcaac caccctctgt actgcccata ctgcgcggga gaagttcttt 2760 tccccgatga gcaaacagaa ttcgcgtggt tgtgtgcgga ttgcaccaga gtttttgaag 2820 tgaaatatca cggccaggac gatccagtgc acaggccagc accagcaaag tccacatcgc 2880 aagcattaaa agaatctctc gaaagacaca aaagaggtga gtcgcaacaa tgagctttca 2940 actagttaac gccctgaaaa atactggttc ggtaaaagat cccgagatct cacccgaagg 3000 acctcgcacg accacaccgt tgtcaccaga ggtagcaaaa cataacgagg aactcgtcga 3060 aaagcatgct gctgcgttgt atgacgccag cgcgcaagag atcctggaat ggacagccga 3120 gcacgcgccg ggcgctattg cagtgacctt gagcatggaa aacaccgtgc tggcggagct 3180 ggctgcgcgg cacctgccgg aagctgattt cctctttttg gacaccggtt accacttcaa 3240 ggagaccctt gaagttgccc gtcaggtaga tgagcgctat tcccagaagc ttgtcaccgc 3300 gctgccgatc ctcaagcgca cggagcagga ttccatttat ggtctcaacc tgtaccgcag 3360 caacccagcg gcgtgctgcc gaatgcgcaa agttgaaccg ctggcggcgt cgttaagccc 3420 atacgctggc tggatcaccg gcctgcgccg cgctgatggc ccaacccgtg ctcaagcccc 3480 tgcgctgagc ttggatgcca ccggcaggct caagatttct ccaattatca cctggtcatt 3540 ggaggaaacc aacgagttca ttgcggacaa caacctcatc gatcacccac ttacccatca 3600 gggttatcca tca 3613 <210> 28 <211> 3311 <212> DNA <213> Corynebacterium glutamicum <400> 28 ctcattccag cgtcacgacg ttccgaaggt actggttacc tggcattggg cactaccgtt 60 tctgcagcac ttggaccagc cctagcactt tttgtcctag gaacatttga ttacgacatg 120 ctgtttatcg tggtcttggc aacctcggtc atctctttga tcgccgtcgt gttcatgtac 180 tttaagacca gcgaccctga gccttctggg gaaccagcca agttcagctt caaatctatt 240 atgaacccaa agatcatccc catcggcatc tttatcttgc ttatttgctt tgcttactct 300 ggcgtcattg cctacatcaa cgcatttgct gaagaacgcg atctgattac gggtgctgga 360 ttgttcttca ttgcctacgc agtatcaatg tttgtgatgc gcagcttcct tggcaaactg 420 caggaccgtc gcggagacaa cgtcgttatt tactttggat tgttcttctt cgttatttcc 480 ttgacgattt tgtcctttgc cacttccaac tggcacgttg tgttgtccgg agtcattgca 540 ggtctgggat acggcacttt gatgccagca gtgcagtcca tcgctgttgg tgtagtagac 600 aaaaccgaat tcggtacggc cttctccact ttgttcctgt ttgtggactt aggttttggc 660 tttggaccta ttatcctggg agcagtttct gcggcaattg gtttcggacc tatgtatgca 720 gcactggcag gtgtgggtgt gattgccgga atcttctacc tgttcacaca cgctcgcacc 780 gatcgagcta agaatggctt tgttaaacac ccagagcctg tcgctttagt tagctagttc 840 tttcagcttt ccctcccgat cagcgtaaac cggcccttcc ggttttgggg tacatcacag 900 aacctgggct agcggtgtag acccgaaaat aaacgagcct tttgtcaggg ttaaggttta 960 ggtatctaag ctaaccaaac accaacaaaa ggctctaccc atgaagtcta ccggcaacat 1020 catcgctgac accatctgcc gcactgcgga actaggactc accatcaccg gcgcttccga 1080 tgcaggtgat tacaccctga tcgaagcaga cgcactcgac tacacctcca cctgcccaga 1140 atgctcccaa cctggggtgt ttcgtcatca cacccaccgg atgctcattg atttacccat 1200 cgtcgggttt cccaccaaac tgtttatccg tctacctcgc taccgctgca ccaaccccac 1260 atgtaagcaa aagtatttcc aagcagaact aagctgcgct gaccacggta aaaaggtcac 1320 ccaccgggtc acccgctgga ttttacaacg ccttgctatt gaccggatga gtgttcacgc 1380 aaccgcgaaa gcacttgggc tagggtggga tttaacctgc caactagccc tcgatatgtg 1440 ccgtgagctg gtctataacg atcctcacca tcttgatgga gtgtatgtca ttggggtgga 1500 tgagcataag tggtcacata atagggctaa gcatggtgat gggtttgtca ccgtgattgt 1560 cgatatgacc gggcatcggt atgactcacg gtgtcctgcc cggttattag atgtcgtccc 1620 aggtcgtagt gctgatgctt tacggtcctg gcttggctcc cgcggtgaac agttccgcaa 1680 tcagatacgg atcgtgtcca tggatggatt ccaaggctac gccacagcaa gtaaagaact 1740 cattccttct gctcgtcgcg tgatggatcc attccatgtt gtgcggcttg ctggtgacaa 1800 gctcaccgcc tgccggcaac gcctccagcg ggagaaatac cagcgtcgtg gtttaagcca 1860 ggatccgttg tataaaaacc ggaagacctt gttgaccacg cacaagtggt tgagtcctcg 1920 tcagcaagaa agcttggagc agttgtgggc gtatgacaaa gactacgggg cgttaaagct 1980 tgcgtggctt gcgtatcagg cgattattga ttgttatcag atgggtaata agcgtgaagc 2040 gaagaagaaa atgcggacca ttattgatca gcttcgggtg ttgaaggggc cgaataagga 2100 actcgcgcag ttgggtcgta gtttgtttaa acgacttggt gatgtgttgg cgtatttcga 2160 tgttggtgtc tccaacggtc cggtcgaagc gatcaacgga cggttggagc atttgcgtgg 2220 gattgctcta ggtttccgta atttgaacca ctacattctg cggtgcctta tccattcagg 2280 gcagttggtc cataagatca atgcactcta aaacaggaag agcccgtaaa cctctgacta 2340 gcgtcaccct ctgattaagg cgaccgcgga tttaagagca gaggctgcca cgagcgcatc 2400 ttcacggctg tgtgttgtac taaaagtaca gcgcacagcc gttcgtgctt gatcctcctc 2460 aagccccaac gccagcaaca catgggatac ctctccggaa ccacaggcag aaccagggga 2520 gcacacaatg ccttggcgtt ccaattccag aagaacagtt tcagatccta tgctgtcgaa 2580 gagaaaagat gcgtgtccat caatgcgcat cctaggatgt ccagtcaggt gtgctcccgg 2640 gatagtgaga acttcctcga tgaattcgcc aagatctgga taggattccg ccctggccaa 2700 ttccaaggca gtggcaaagg cgatagcccc cgcaacgttt tccgtgccac tacgccgccc 2760 tttttcctgg ccgccgccat ggattaccgg ctccagggga agctttgacc ataacactcc 2820 aatcccttta ggcgcaccga atttatgacc cgacaaactt aacgcgtcaa ctcccaagtc 2880 aaaggttaaa tgtgcagctt gcactgcatc ggtgtgaaaa ggcgtactgc ttaccgccgc 2940 caactcagct atcggctgaa tggttcccac ctcattgttg gcataaccaa tgctgatcaa 3000 tgtggtgtcc ggcctgactg ctttgcggag accctccggg gagatcagcc cagtgtgatc 3060 gggggatagg taggtgatct cgaaatcatg aaacctttca agataagcag cagtttctag 3120 gacactgtca tgctcgatcg gggtggtgat gaggtgccgg ccacgaggat tagctaagca 3180 cgctcctttg atagcgaggt tgttggcttc tgatccaccc gacgtaaacg tcacctgtgt 3240 ggggcgtcct ccgataatgc gggccacccg agttcgagca tcctccagcc ccgcagaggc 3300 gagtcttccc a 3311 <210> 29 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 29 acccggggat cctctagaat gtttgtgatg cgcag 35 <210> 30 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 30 gtcagagagt acttacgctg atcgggaggg aaagc 35 <210> 31 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 31 atcagcgtaa gtactctctg actagcgtca ccctc 35 <210> 32 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 32 ctgcaggtcg actctagaaa agggattgga gtgtt 35 <210> 33 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 33 caacgaaagg aaacaatgtc tacttcagtt acttc 35 <210> 34 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 34 tagtcagaga gtgatttaga cgttaaagta ctttg 35 <210> 35 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 35 atcaaaacag atatcatgac aacaaccacc ggaag 35 <210> 36 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 36 cgctagtcag agagttcaca ccaaatcttc ctcag 35 <210> 37 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 37 ccgatcagcg taagtagaaa catcccagcg ctact 35 <210> 38 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 38 aactgaagta gacattgttt cctttcgttg ggtac 35 <210> 39 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 39 tactttaacg tctaaggtac cggcgcttca tgtca 35 <210> 40 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 40 ggtggttgtt gtcatgatat ctgttttgat ctcct 35 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 41 atccccatcg gcatctttat 20 <210> 42 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 42 cgatcacact gggctgatct 20 <210> 43 <211> 381 <212> PRT <213> Corynebacterium glutamicum <400> 43 Met Thr Leu Thr Phe His Trp Phe Leu Ser Thr Ser Gly Asp Ser Arg 1 5 10 15 Gly Ile Ile Gly Gly Gly His Gly Ala Glu Lys Ser Gly Thr Ser Arg 20 25 30 Glu Leu Ser His Ser Tyr Leu Lys Gln Leu Ala Leu Ala Ala Glu Thr 35 40 45 Asn Gly Phe Glu Ser Val Leu Thr Pro Thr Gly Thr Trp Cys Glu Asp 50 55 60 Ala Trp Ile Thr Asp Ala Ser Leu Ile Glu Ala Thr Lys Arg Leu Lys 65 70 75 80 Phe Leu Val Ala Leu Arg Pro Gly Gln Ile Gly Pro Thr Leu Ser Ala 85 90 95 Gln Met Ala Ser Thr Phe Gln Arg Leu Ser Gly Asn Arg Leu Leu Ile 100 105 110 Asn Val Val Thr Gly Gly Glu Asp Ala Glu Gln Arg Ala Phe Gly Asp 115 120 125 Phe Leu Asn Lys Glu Glu Arg Tyr Ala Arg Thr Gly Glu Phe Leu Asp 130 135 140 Ile Val Ser Arg Leu Trp Arg Gly Glu Thr Val Thr His His Gly Glu 145 150 155 160 His Leu Gln Val Glu Gln Ala Ser Leu Ala His Pro Pro Glu Ile Ile 165 170 175 Pro Glu Ile Leu Phe Gly Gly Ser Ser Pro Ala Ala Gly Glu Val Ala 180 185 190 Ala Arg Tyr Ala Asp Thr Tyr Leu Thr Trp Gly Glu Thr Pro Asp Gln 195 200 205 Val Ala Gln Lys Ile Asn Trp Ile Asn Glu Leu Ala Ala Gln Arg Gly 210 215 220 Arg Glu Leu Arg His Gly Ile Arg Phe His Val Ile Thr Arg Asp Thr 225 230 235 240 Ser Glu Glu Ala Trp Val Val Ala Glu Lys Leu Ile Ser Gly Val Thr 245 250 255 Pro Glu Gln Val Ala Lys Ala Gln Ala Gly Phe Ala Thr Ser Lys Ser 260 265 270 Glu Gly Gln Arg Arg Met Ala Glu Leu His Ser Lys Gly Arg Ala Phe 275 280 285 Thr Ser Gly Ser Thr Ala Arg Asp Leu Glu Val Tyr Pro Asn Val Trp 290 295 300 Ala Gly Val Gly Leu Leu Arg Gly Gly Ala Gly Thr Ala Leu Val Gly 305 310 315 320 Ser His Glu Glu Val Ala Asp Arg Ile Glu Glu Tyr Ala Ala Leu Gly 325 330 335 Leu Asp Gln Phe Val Leu Ser Gly Tyr Pro Asn Leu Glu Glu Ala Phe 340 345 350 His Phe Gly Glu Gly Val Ile Pro Glu Leu Leu Arg Arg Gly Val Asp 355 360 365 Ile Lys Asn Gln Glu Ser Arg Val Leu Glu Pro Val Gly 370 375 380

Claims (11)

A method for producing a sulfur-containing amino acid or a sulfur-containing amino acid derivative, comprising culturing a microorganism whose protein activity encoded by the ssuD gene is enhanced compared to the intrinsic activity in a medium containing thiosulfate.
The method of claim 1, wherein the protein encoded by the ssuD gene has a thiosulfate reductase activity, a sulfur-containing amino acid or a sulfur-containing amino acid derivative.
The method of claim 1, wherein the protein encoded by the ssuD gene comprises an amino acid sequence having at least 80% homology with SEQ ID NO: 43.
The method of claim 1, wherein the microorganism is a microorganism of the genus Corynebacterium sp. or Escherichia sp.
The method of claim 4, wherein the microorganisms are Corynebacterium glutamicum , Corynebacterium callunae , Corynebacterium deserti , Corynebacterium crenatum ( Corynebacterium crenatum ) or Escherichia coli ( Escherichia coli ) that, the manufacturing method.
The method of claim 1, wherein the method comprises recovering a sulfur-containing amino acid or a sulfur-containing amino acid derivative from the microorganism or culture medium.
The method of claim 1, wherein the reinforcement of the protein activity encoded by the ssuD gene is achieved by the copy number increased, the promoter activity enhancement, initiation codon replacement or combination thereof, of the polynucleotide encoding the protein encoded by the ssuD gene That, the manufacturing method.
The method of claim 1, wherein the sulfur-containing amino acid or sulfur-containing amino acid derivative is methionine, cysteine, cystine, lanthionine, homocysteine, homocystine, homocysteine, and homocysteine. Lanthionine (homolanthionine), taurine (taurine) that is any one or more selected from, the manufacturing method.
A microorganism that produces a sulfur-containing amino acid or a sulfur-containing amino acid derivative in which the protein activity encoded by the ssuD gene is enhanced compared to the intrinsic activity.
The microorganism according to claim 9, wherein the microorganism produces a sulfur-containing amino acid or a sulfur-containing amino acid derivative by using thiosulfate as a sulfur source.
a microorganism, or a culture thereof, in which the protein activity encoded by the ssuD gene is enhanced compared to the intrinsic activity; And a composition for preparing a sulfur-containing amino acid or a sulfur-containing amino acid derivative comprising thiosulfate.